Apoptosis associated protein Bbk

ABSTRACT

The present invention is directed to an isolated Bbk protein, nucleotide sequences coding for and regulating expression of the protein, antibodies directed against the protein, and recombinant vectors and host cells containing the genetic sequences coding for and regulating the expression of the protein sequence. The invention is also directed to genomic DNA, cDNA, and RNA encoding the Bbk protein sequence and to corresponding antisense RNA sequences. Antibodies can be used to detect Bbk in biological specimens, including, for example, human tissue samples. The present invention is further directed to methods of treating degenerative disorders characterized in inappropriate cell proliferation or inappropriate cell death. The present invention is further directed to methods for diagnosing degenerative disorders characterized in inappropriate cell proliferation or inappropriate cell death, as well as methods for monitoring the progress of such degenerative disorders.

FIELD OF THE INVENTION

The present invention relates generally to the field of cell physiology,and more particularly, to apoptosis. Even more particularly, the presentinvention is related to the novel apoptosis associated protein Bbk; tonucleotide sequences encoding Bbk; to products and processes involved inthe cloning, preparation and expression of genes and nucleotidesequences encoding Bbk; to antibodies with specificity to Bbk; and todiagnostic and therapeutic uses of the above.

BACKGROUND OF THE INVENTION

"Apoptosis" refers to cell suicide that proceeds by an active,physiological process (Kerr, J. F., et al., Br. J. Cancer 26:239-257(1972); Wyllie, A. H., Nature 284:555-556 (1980)). Cells that die byapoptosis undergo characteristic morphological changes, including cellshrinkage, and nuclear condensation and fragmentation. Apoptosis playsan important role in developmental processes, including morphogenesis,maturation of the immune system, and tissue homeostasis whereby cellnumbers are limited in tissues that are continually renewed by celldivision (Ellis, R. E., et al., Annu. Rev. Cell. Biol.7:663-698 (1991);Oppenheim, R. W., et al., Neurosci. 14:453-501 (1991); Cohen, J. J., etal., Annul. Rev. Imnnitnol. 10:267-293 (1992); Raff, M. C., Nature356:397-400 (1992)). Apoptosis is an important cellular safeguardagainst tumorigenesis (Williams, G. T., Cell 65:1097-1098 (1991); Lane,D. P. Nature 362:786-787 (1993)). Defects in the apoptotic pathway maycontribute to the onset or progression of malignancies. Under certainconditions, cells undergo apoptosis in response to forced expression ofoncogenes, or other genes that drive cell proliferation; (Askew, D., etal., Oncogene 6:195-1922 (1991); Evan, G. I., et al., Cell 69:119-128(1992); Rao, L., et al., Proc. Natl. Acad Sci. USA 89:7742-7746 (1992);Smeyne, R. J., et al., Nature 363:166-169 (1993)). A variety ofdegenerative disorders may involve aberrant apoptosis, resulting inpremature or inappropriate cell death (Barr, P. J., et al.,Biotechnology 12:487-493 (1994)). Productive infection by certainviruses may depend on suppression of host cell death by anti-apoptoticviral gene products (Rao, L., et al., Proc. Natl. Acad. Sci. USA89:7742-7746 (1992); Ray, C. A., et al., Cell 69:597-604 (1992); White,E., et al., Mol. Cell. Biol. 12:2570-2580 (1992); Vaux, D. L., et al.,Cell 76:777-779 (1994), and inhibition of apoptosis can alter the course(i.e. lytic vs. latent) of viral infection; (Levine, B., et al., Nature361:739-742 (1993)). Widespread apoptosis of T lymphocytes triggered byHIV infection may, at least in part, be responsible for the immunesystem failure associated with AIDS (Gougeon M., et al., Science260:1269-1270 (1993)). The roles of apoptosis in normal and pathologicalcell cycle events are reviewed in Holbrook, N.J., et al., Eds., CellularAging and Cell Death, Wiley-Liss, Inc., Publisher, New York, N.Y.(1996).

The bcl-2 gene product has been intensively studied as a potentsuppressor of apoptotic cell death. The bcl-2 gene was originallyidentified at the t(14:18) translocation breakpoint that occursfrequently in human B cell follicular lymphomas (Bakhshi, A., et al.,Cell 41:899-906 (1985); Cleary, M. L., et al., Proc. Natl. Acad. Sci.USA 82: (1985); Tsujimoto, Y., et al., Science 229:1390-1393 (1985)).This translocation results in the constitutive activation of bcl-2 geneexpression due to juxtaposition with the immunoglobulin heavy chainlocus. Bcl-2 functions as an oncogene in this disease by inappropriatelysuppressing apoptosis that would normally limit the accumulation ofthese cells (McDonnell, T. J., et al., Cell 57:79-88 (1989); Hockenbery,D., et cal., Nature 348:334-336 (1990)). Consequently, B cellsaccumulate during the indolent stage of the lymphoma due to theirfailure to die rather than by uncontrolled proliferation.

The anti-apoptotic activity of Bcl-2 is not restricted to B cells. Alarge number of studies have demonstrated that ectopic Bcl-2 expressioncan suppress apoptosis triggered by diverse stimuli in a multitude ofcell lineages (Vaux, D. L., et al., Nature 335:440-442 (1988); Sentman,C. L., et al., Cell 67:879-888 (1991); Strasser, A., et al., Cell67:889-899 (1991); Hockenbery, D. M., et al., Cell 75:241-251 (1993)).Bcl-2 blocks cell death induced by growth factor withdrawal, DNA damage,oncogene expression, oxidative stress, and viral infection. The abilityof Bcl-2 to block apoptosis in virtually every system suggests thatBcl-2 is closely connected with the machinery that actually carries outthe death program. This view is further supported by the conservation ofBcl-2 function across species. The ced9 gene in the nematode C. elegansfunctions to suppress programmed death in certain cell lineages of thedeveloping worm (Ellis, H. M., et al, Cell 44:817-829 (1986)). Ced9appears to be a functional homologue of Bcl-2, since Bcl-2 cancomplement ced9 in transgenic worms (Vaux, D. L., et al., Science258:1955-1957 (1991)). Bcl-2 can also function in insect cells asdemonstrated by the ability of Bcl-2 to suppress apoptosis induced byBaculovirus infection (Alnemri, E. S., et al., Proc. Natl. Acad. Sci.USA 89:7295-7299 (1992)). The molecular mechanism whereby Bcl-2 operatesto block cell death is poorly understood.

Additional cellular genes that exhibit significant sequence homologywith Bcl-2 have been identified and, where tested, these genes appearalso to function as regulators of apoptotic cell death. One Bcl-2relative, Bcl-X, was isolated by low stringency DNA hybridization to theBcl-2 gene (Boise, L. H., et al., Cell 74:597-608 (1993)). The Bcl-X RNAis differentially spliced to produce a long form, termed Bcl-X_(L), anda shorter forn, Bcl-X_(S), bearing a short internal deletion. Bcl-X_(L),functions to suppress cell death, much like Bcl-2, whereas the deletedform, Bcl-X_(S), can inhibit protection by Bcl-2 and may function as a"dominant negative" species. A second Bcl-2 relative, Bax, wasidentified biochemically as protein found in co-immunoprecipitates withBcl-2 (Oltvai, Z. N., et al., Cell 74:609-619 (1993)). Isolation of thecorresponding cDNA revealed that the Bax protein shows substantialsequence homology to Bcl-2. Bax forms heterodimers with Bcl-2 andappears to induce apoptosis and function as a negative regulator ofBcl-2 function. Ectopic expression of Bax was shown to block theprotection against apoptosis afforded by Bcl-2 expression.

Two additional cellular Bcl-2 relatives, Mcl-1 and Al(Kozopas, K. M., etal., Proc. Natl. Acad. Sci. USA 90:3516-3520 (1993); Lin, E. Y., et al.,J. Immunol. 1.51:1979-1988 (1993)) were originally isolated as mRNAsinduced in response to specific stimuli: phorbol ester induceddifferentiation of mycloid leukemia cells (Mcl-1); and GM-CSF treatmentof murine bone marrow cells (A1). It is not yet known whether eitherMcl-1 or A1 can modulate apoptosis.

In addition to these cellular Bcl-2 relatives, a number of Bcl-2homologues encoded by DNA viruses have been identified. The Epstein-Barrvirus BHRF-1 gene product was noted to contain sequence homology toBcl-2 and has subsequently been shown to function as a suppressor ofapoptosis (Henderson, et al., Proc. Natl. Acad. Sci. USA 90:8479-8488(1993)). Likewise, the African swine fever virus LMW5-HL gene encodes aprotein structurally similar to Bcl-2 (Neilan, J. G., et al., J. Virol.67:4391-4394 (1993)). The Adenovirus E1b 19kD protein appears to befunctionally equivalent to Bcl-2, although the primary sequence homologyis quite limited (White, E., et al., Mol. Cell. Biol. 12:2570-2580(1992)). It is likely that these genes function to ensure replication ofviral DNA by preventing apoptosis of the infected cell. The finding thatunrelated DNA viruses have evolved genes that apparently function tomimic the action of Bcl-2, supports the conclusion that Bcl-2 representsan important apoptosis regulator.

The isolation and characterization of a bcl-2 related gene, termed bak,is described in co-pending U.S. application Ser. No. 08/321,071, filed11 Oct., 1994, U.S. Pat. No. 5,672,686 which is a continuation-in-partof U.S. application Ser. No. 8/287,427, filed 9 Aug., 1994, abandoned(bak is referred to therein as bcl-y), the disclosures of which areincorporated herein by reference. Ectopic Bak expression accelerates thedeath of an IL-3 dependent cell line upon cytokine withdrawal, andopposes the protection against apoptosis afforded by Bcl-2. In addition,enforced expression of Bak is sufficient to induce apoptosis of serumdeprived fibroblasts, raising the possibility that Bak directlyactivates, or is itself a component of, the cell death machinery.

Known cellular Bcl-2 related genes, where analyzed, have distinctpatterns of expression and thus may function in different tissues. Thecell death program is in place in all tissues and may be regulated bydifferent Bcl-2 related genes. While Bcl-2 expression is required formaintenance of the mature immune system, it is desirable to identifyother genes which may govern apoptotic cell death in other lineages.From the perspective of pharmaceutical development, it would bedesirable to identify or develop agents that either activate or suppressapoptosis, depending on the clinical setting.

SUMMARY OF THE INVENTION

The present inventor has surprisingly discovered a novel composition ofmatter which has been isolated and characterized, and which is describedin a number of embodiments herein. Referred to herein as "Bbk," itappears to be a member of the Bcl-2 family, and can, inter alia, induceapoptosis in cells and oppose the function of Bcl-2 and related celldeath suppresors in cells. Isolation of a full length human Bbk cDNArevealed that the deduced Bbk amino acid sequence shares homology withBcl-2. Bbk mRNA appears to be widely expressed in primary human tissues.Bbk is an important regulator of apoptosis in human tissues and/or tumorcells.

Expression of Bbk accelerates apoptosis when expressed in normal ratfibroblasts (Rat1), and in human tumor cell lines including HeLa andBT549 cells. The co-expression of Bak and Bbk in Rat 1 cells does notblock the induction of apoptosis, suggesting that their ability to bindeach other does not inhibit their ability to promote apoptosis. Theircoexpression may result in cooperative induction of cell death. Theapoptotic function of Bbk can be reversed by the coexpression of theknown survival proteins, Bcl-2, Bcl-x_(L) and Epstein-Barr virus BHRF 1.Increasing the ratio of Bbk relative to the survival proteins mayrestore apoptosis as has been previously shown with the apoptosispromoting protein Bik.

The present invention thus relates to an apoptosis associated proteinBbk, products and processes involved in the cloning, preparation andexpression of genes for Bbk; antibodies with specificity to Bbk; andnucleotide probes corresponding to the Bbk nucleotide sequence orportions thereof. The Bbk polypeptide is useful for producing antibodiesthereto. The antibodies and probes are useful for detecting andisolating Bbk in biological specimens including for example, cells fromall human tissues including heart tissue, lung tissue, tumor cells,placenta, liver, skeletal muscle, kidney, and pancreas.

The present invention further relates to species homologs and viralhomologs of Bbk.

The present invention relates to the identification, characterizationand sequencing of cDNAs and genomic fragments which encode the Bbk thatis present in human cells.

According to the present invention, there are provided genetic sequencesencoding Bbk. The instant invention also provides for expression vectorscontaining such genetic sequences, hosts transformed with suchexpression vectors, and methods for producing the genetically engineeredor recombinant Bbk.

The present invention also provides antibodies which specificallyrecognize Bbk.

The Bbk cDNA and recombinant protein are useful for making antibodieswhich specifically recognize Bbk. Such antibodies are useful fordetecting and isolating Bbk in a biological specimen. The present Bbkprotein is also useful as a regulator of apoptosis.

A small cDNA from an EBV-transformed B-cell line has been isolated. Theamino acid sequence of the Bbk protein shares sequence homology withBcl-2 domains.

The present invention further relates to a method for isolating Bbkpartial clones using polymerase chain reaction (PCR) cloning, fromdiverse human tumor cell lines.

The present invention is further directed to methods for inducing orsuppressing apoptosis in individuals suffering from degenerativedisorders characterized by inappropriate cell proliferation orinappropriate cell death, respectively. Degenerative disorderscharacterized by inappropriate cell proliferation include, for example,inflammatory conditions, cancer, including lymphomas, genotypic tumors,etc. Degenerative disorders characterized by inappropriate cell deathinclude, for example, autoimmune diseases, acquired immunodeficiencydisease (AIDS), cell death due to radiation therapy or chemotherapy,etc.

The present invention also relates to methods for detecting the presenceof Bbk protein, as well as methods directed to the diagnosis ofdegenerative disorders, which disorders are associated with an increasedor decreased level of expression or mutations of Bbk, as compared to theexpected level of Bbk expression in the normal cell population.

The present invention is further directed to methods for monitoring theprogress of degenerative disorders associated with increased ordecreased levels of expression of Bbk, by monitoring Bbk expression.

The present invention also relates to methods for determining whether adisease/degenerative disorder is linked to abnormal Bbk expression, aswell as methods for determining the effect of over-expression or loss ofexpression of Bbk in animal models such as transgenic mice and/orhomozygous null mice. Methods for determining whether adisease/degenerative disorder is linked to abnormal Bbk expressioninclude analyzing Bbk expression in diseased tissue as compared tonormal tissue by for example, Northern and/or Western blots, as well asby other assay methods readily chosen and employed by those of ordinaryskill in the art.

The present invention relates to hybrids of Bbk for therapeutic use.

The present invention also relates to methods for modulating apoptoticeffects by administering the present Bbk protein, mutant protein orhybrids to an individual suffering from a degenerative disordercharacterized by inappropriate cell proliferation or inappropriate celldeath in order to stabilize inappropriate cell proliferation (i.e.,induce apoptosis) or stabilize inappropriate cell death (i.e., suppressapoptosis), respectively, and/or in either case to restore normal cellbehavior. FIG. 3 illustrates levels of Bbk mRNA expressed in a varietyof healthy fetal and adult tissues.

The present invention further relates to functional equivalentsincluding functional fragments of Bbk including, for example, peptidesof Bbk such as BH1 and BH2, and other regions of homology recognized bythe present inventor between Bbk and other apoptosis related proteinsincluding Bcl-2 and Bax.

In a particular aspect, the invention is directed to a novel proteindomain which has been identified and mapped to a short subsequence inthe central portion of the Bbk molecule. This novel protein domain,which the inventor has designated the "Bbk BH3 domain," is essentialboth to Bbk's interaction with Bak, and to Bbk's cell killing function.Truncated Bbk species encompassing the Bbk BH3 domain are themselvessufficient to kill cells in transfection assays.

The Bbk BH3 domain shares less than twenty-live percent identity withthe GD Domain first described in Bak in U.S. application Ser. No.08/440,391, filed 12 May, 1995, U.S. Pat. No. 5,656,725 (BH3 is referredto therein as the GD domain). However, as observed with respect to theGD Domain in Bak, mutation of Bbk BH3 domain elements in Bbk diminishescell killing and protein binding function. Thus, the Bbk BH3 domain isresponsible for mediating key protein/protein interactions ofsignificance to the actions of multiple cell death regulatory molecules.

In one aspect, then, the invention is directed to purified and isolatedpeptides comprising the Bbk BH3 domain and to molecules that mimic itsstructure and/or function, useful for inducing or modulating theapoptotic state of a cell. Chemical compounds that disrupt the functionof the Bbk BH3 domain have utility as apoptosis-modulating agents.Accordingly, in another aspect, the invention is directed to agentscapable of disrupting Bbk BH3 domain function. Such agents include, butare not limited to, molecules that bind to the Bbk BH3 domain, moleculesthat interfere with the interaction of the Bbk BH3 domain with otherprotein(s), and molecules comprising the Bbk BH3 domain which is alteredin some manner. The invention provides methods to identify moleculesthat modulate apoptosis by disrupting the function of the Bbk BH3domain, which accordingly comprise additional contemplated embodiments.

In additional aspects, the present invention relates to products andprocesses involved in the cloning, preparation and expression ofpeptides comprising the Bbk BH3 domain; antibodies with specificity tothe Bbk BH3 domain; and nucleotide sequences encoding the Bbk BH3 domainor portions thereof. Peptides comprising the Bbk BH3 domain are usefulfor producing antibodies thereto. Such antibodies are useful fordetecting and isolating proteins comprising the Bbk BH3 domain inbiological specimens including, for example, cells from all humantissues including heart tissue, lung tissue, tumor cells, brain tissue,placenta, liver, skeletal muscle, kidney, and pancreas, as well as formodulating the apoptotic activity of proteins comprising the Bbk BH3domain in and from such biological specimens, and constitute additionalaspects of the invention.

In yet another aspect, the invention provides for expression vectorscontaining genetic sequences, hosts transformed with such expressionvectors, and methods for producing the recombinant Bbk BH3 domainpeptides of the invention.

The present invention is further directed to methods for inducing orsuppressing apoptosis in the cells and/or tissues of individualssuffering from degenerative disorders characterized by inappropriatecell proliferation or inappropriate cell death, respectively.Degenerative disorders characterized by inappropriate cell proliferationinclude, for example, inflammatory conditions, cancer, includinglymphomas, such as prostate hyperplasia, genotypic tumors, etc.Degenerative disorders characterized by inappropriate cell deathinclude, for example, autoimmune diseases, acquired immunodeficiencydisease (AIDS), cell death due to radiation therapy or chemotherapy,neurodegenerative diseases, such as Alzheimer's disease and Parkinson'sdisease, etc.

The present invention also relates to methods for detecting the presenceof the Bbk BH3 domain peptide, as well as methods directed to thediagnosis of degenerative disorders, which disorders are associated withan increased or decreased level of expression of proteins comprising theBbk BH3 domain, as compared to the expected level of expression of suchproteins in the normal cell population.

The present invention relates to the therapeutic use of peptidescomprising the Bbk BH3 domain.

The present invention also relates to methods for modulating theapoptotic state of a cell by administering peptides comprising the BbkBH3 domain peptide, or mutants thereof, to an individual suffering froma degenerative disorder characterized by inappropriate cellproliferation or inappropriate cell death, in order to stabilizeinappropriate cell proliferation (i.e., induce apoptosis) or stabilizeinappropriate cell death (i.e., suppress apoptosis), respectively,and/or in either case to restore normal cell behavior.

The present invention is also directed to nucleotide probes which can beused to determine the presence of Bbk as well as to identify and isolatehomologs including species homologs and viral homologs.

These and other objects and aspects of the invention will be apparent tothose of skill from the description which follows.

DESCRIPTION OF THE FIGURES

FIGS. 1A-1C. Features of the yeast two-hybrid system (adapted fromClontech manual).

FIG. 1(A). A schematic illustration of the yeast GAL4 protein showingthe DNA binding domain (bd) that interacts with the GAL1 upstreamactivating sequence (UAS) and the transcription activation domain (ad)that stimulates transcription.

FIG. 1(B). The GAL4 bd fused to protein X can bind to the GAL1 UAS butcannot stimulate transcription due to the lack of an activation domain.The GAL4 ad fused to protein Y also fails to stimulate transcription dueto failure to localize to the promoter.

FIG. 1(C). The interaction of GAL4 bd/protein X fusion with the GAL1 UASand its additional interaction with the GAL4 ad/protein Y fusion allowsthe reconstitution of GAL4 function and the stimulation oftranscription.

FIG. 2. The nucleotide sequence and putative open reading frame (ORF) ofclone Bbk SEQ ID NOS:9-11! Arrows indicate the start points of severalrelated clones also isolated by the two-hybrid analysis. The position ofan AAG insert identified in several clones is also indicated.

FIG. 3. Northern blot analysis of human fetal and adult tissues(Clontech). The blots were hybridized with ³² P-labeled Bbk DNA (toppanel) and β-actin DNA as a control. Size markers are as defined by themanufacturer.

FIGS. 4A-4B. Expression of Bbk protein.

FIG. 4(A). In vitro translation of ³⁵ S-labeled Bbk in rabbitreticulocyte lysate. Controls include translation of luciferase proteinwas resolved by SDS gel electrophoresis and visualized byautoradiography. The gel mobilities of pre-stained protein molecularweight markers (Amersham) are shown.

FIG. 4(B). Expression of Bbk in transfected cells. Plasmids expressingthe hemagglutinin (HA) epitope-tagged Bax or Bbk were transfected intoCOS7 cells. Lysates were prepared 48 hrs after transfection. Lysates ofuntransfected COS7 cells are included as a negative control. Proteinswere detected by SDS gel electrophoresis and Western blot of celllysates with the anti-HA monoclonal antibody 12CA5 (BoehringerMannheim). The Bax and Bbk proteins are indicated with arrows.

FIGS. 5A-5B. Effect of Bbk expression on viability of Rat1 cells. Cellswere transfected with plasmids as indicated with a plasmid expressingβ-galactosidase (pRcCMV/βgal, 0.16 μg). Cells were stained 24 hrspost-transfection to identify live and dead β-galactosidase expressingcells. Values from triplicate experiments were averaged and plotted witherror bars representing SEM.

FIG. 5(A). Effect of transient expression of vector (0.42 μg pRcCMV),Bak (0.21 μg pcDNA1/HABak+0.21 μg pRcCMV), Bbk (0.21 μgpcDNA3/HABbk+0.21 μg pRcCMV), or Bak+Bbk (0.21 μg pcDNA3/HABak+0.21 μgpcDNA3/HABbk) in Rat1 cells.

FIG. 5(B). Effect of the survival proteins Bcl-2 (0.21 μgpcDNA3/HABbk+0.21 μg pRcCMV/Bcl-2), Bcl-x_(L) (0.21 μg pcDNA3/HABbk+0.21μg pRcCMV/Bcl-x_(L)), or Epstein Barr virus BHRF1 (0.21 μgpcDNA3/HABbk+0.21 μg pRcCMV/BHRF1) upon Bbk induction of apoptosis inRat1 cells.

FIGS. 6A-6B. Effect of Bbk expression on viability of HeLa cells andBT549 cells.

FIG. 6(A). HeLa cells were transfected with pRcCMV/βgal (0.16 μg) plusvector (0.42 μg pRcCMV), Bak (0.21 μg pcDNA1/HABak+0.21 μg pRcvCMV), orBbk (0.21 μg pcDNA3/HABbk+0.21 μg pRcCMV). Stained cells were scored andplotted as described in FIG. 5.

FIG. 6(B). BT549 cells were transfected as described in panel (A).

FIG. 7. Interaction of Bbk with Bcl-2 family members. A glutathioneS-transferase (GST) fusion protein of Bbk was produced in E. coli andpurified on glutathione agarose. Purified Bbk or GST control protein wasincubated with ³⁵ S labeled in vitro translated (IVT) HA-tagged Bak,Bax, Bik or Flag-tagged Bcl-x_(L). Complexes were captured onglutathione agarose beads, subjected to SDS gel elctrophoresis, andvisualized by autoradiograhy. Captured complexes are compared to analiquot of the input IVT material.

FIGS. 8A-8B. Sequence alignment of Bbk with Bcl-2 family member BH2 andBH3 domains.

FIG. 8(A). The BH2 domain sequences of Bak SEQ ID NO:13!, Bax SEQ IDNO:14!, Bik SEQ ID NO:15!, Bcl-2 SEQ ID NO:16! and Bcl-x_(L) SEQ IDNO:17! are aligned with the homologous regions of Bbk SEQ ID NO:12!.Residues that are identical or conservative in at least three of theproteins are boxed. Black boxes indicate identical residues while greyboxes indicate conservative residues. Numbering indicates the positionof the first amino acid residue shown for each sequence.

FIG. 8(B). The BH3 domain sequences of Bak SEQ ID NO:19!, Bax SEQ IDNO:20!, Bik SEQ ID NO:21!, Bcl-2 SEQ ID NO:22! and Bcl-x_(L) SEQ IDNO:233! are aligned with the homologous regions of Bbk SEQ ID NO:18!.Shading and numbering are as described in panel (A).

FIGS. 9A-9C. Deletion and point mutation analysis of Bbk in Rat1 cells.

FIG. 9(A). Rat1 cells were transfected with pRcCMV/βgal (0.16 μg) plusvector (0.42 μg pRcCMV), full length Bbk (0.42 μg pcDNA3/HABbk), ordeletion mutants of Bbk (0.42 μg pcDNA3/HAΔ1-105 or 0.42 μgpcDNA3/HAΔ142-249). Stained cells were scored and plotted as describedin FIG. 5.

FIG. 9(B). Alanine point mutants of the Bbk BH3 domain (PM-LVLEE SEQ IDNO:25!, PM-V SEQ ID NO:26!, PM-L SEQ ID NO:27!, PM-EE SEC ID NO:28!) arecompared to the wild type Bbk BH3 domain SEQ ID NO 24!. The shading isas described in FIG. 8 with Alanine substitutions indicated as outlinedboxes.

FIG. 9(C). The alanine point mutants shown in panel (B) (0.42 μg eachpcDNA3/HAPM-LVLEE, pcDNA3/HAPM-V, pcDNA3/HAPM-L, pcDNA3/HAPM-EE plus0.16 μg pRcCMV/βgal) were transfected into Rat1 cells and compared tocells transfected with vector control plasmid or wild type Bbk asdescribed in panel (A). Stained cells were scored and plotted asdescribed in FIG. 5.

FIG. 10. Effect of Bbk BH3 domain expression on the viability of Rat1cells. Rat1 cells were transfected with pRcCMV/bβgal (0.16 mg) plusvector (0.42 mg pRcCMV), full length Bbk (0.42 mg pRcCMV/HABbk), or BbkBH3 domain (0.42 mg pRcCMV/HABbkBH3). Stained cells were scored andplotted as described in FIG. 5.

FIG. 11. Interaction of Bbk point mutants with Bak analyzed by yeasttwo-hybrid system. Bak bait plasmid (pAS2/BakΔC) was co-transformed intoyeast with plasmids expressing Gal4 activation domain fusions of alaninepoint mutations of Bbk (pACF/PM-LVLEE, pACT/PM-V, pACT/PM-L, andpACT/PM-EE) and wild type Bbk (pACT/Bbk). As a positive control,plasmids supplied by the manufacturer (Clontech) expressing p-53 bait(pVA3) and SV40 T antigen (pTD1) were co-transformed as above. For anegative control, pACT/Bbk was co-transformed with pAS2, which expressesonly the Gal4 activation domain as bait. Three individual colonies fromeach transformation were analyzed in triplicate for β-galactosidaseactivity using the liquid culture assay described by the manufacturer(Clontech). The mean of triplicate measurements from each of the threecolonies were then averaged to generate a single value for each pair ofinteracting proteins. The data are plotted as units of β-galactosidase(Miller, J. H., Experiments in Molecular Genetics, Cold Spring HarborLaboratory Press, Planview, N.Y. (1972)) with error bars representingthe SEM.

DETAILED DESCRIPTION OF THE INVENTION

Technical and scientitic terms used herein have the meanings commonlyunderstood by one of ordinary skill in the art to which the presentinvention pertains, unless otherwise defined. Reference is made hereinto various methodologies known to those of skill in the art.Publications and other materials setting forth such known methodologiesto which reference is made are incorporated herein by reference in theirentireties as though set forth in full. Standard reterence works settingforth the general principles of recombinant DNA technology includeSambrook, J., et al., Molecular Cloning,: A Laboratory Manual, 2d Ed.,Cold Spring Harbor Laboratory Press, Planview, N.Y. (1989); McPherson,M. J., Ed., Directed Mutagenesis: A Practical Approach, IRL Press,Oxford (1991); Jones, J., Amino Acid and Peptide Synthesis, OxfordScience Publications, Oxford (1992); Austen, B. M. and Westwood, O. M.R., Protein Targeting and Secretion, IRL Press, Oxford (1991). Anysuitable materials and/or methods known to those of skill can beutilized in carrying out the present invention; however, preferredmaterials and/or methods are described. Materials, reagents and the liketo which reference is made in the following description and examples areobtainable from commercial sources, unless otherwise noted.

This invention is directed most generally to a novel protein designated"Bak binding killer" protein or "Bbk" based upon its ability to bindspecifically to the protein Bak and to kill immortalized human tumorcells. Accordingly, this invention comprises amino acid sequences of Bbkor Bbk mutants, genetic sequences coding for such amino acid sequences,expression vehicles containing the genetic sequences, hosts transformedtherewith and recombinant Bbk and antisense RNA produced by suchtransformed host expression. The invention further comprises antibodiesdirected against Bbk and/or fragments thereof or against Bbk mutants.

The process for genetically engineering such protein sequences,according to the invention, is facilitated through the cloning ofgenetic sequences which are capable of encoding the peptide and throughthe expression of such genetic sequences. As used herein, the term"genetic sequences" is intended to refer to a nucleic acid molecule(preferably DNA). Genetic sequences which are capable of encoding theproteins are derived from a variety of sources. These sources includegenomic DNA, cDNA, synthetic DNA, and combinations thereof. Thepreferred source of the genomic DNA or mRNA is human tissue includingheart, lung, tumor cells, placenta, liver, skeletal muscle, andpancreas. The mRNA may then be used to obtain cDNA by techniques knownto those skilled in the art. Probes may be synthesized based on thenucleotide sequence of Bbk by methods known in the art.

The Bbk protein or fragment genomic DNA of the invention may or may notinclude naturally occurring introns. Moreover, such genomic DNA may beobtained in association with the 5' promoter region of the Bbk proteingene sequences and/or with the 3' transcriptional termination region.Further, such genomic DNA may be obtained in association with thegenetic sequences which encode the 5' non-translated region of the Bbkprotein mRNA and/or with the genetic sequences which encode the 3'non-translated region. To the extent that a host cell can recognize thetranscriptional and/or translational regulatory signals associated withthe expression of the mRNA and protein, the 5' and/or 3' non-transcribedregions of the native gene, and/or the 5' and/or 3' non-translatedregions of the mRNA, may be retained and employed for transcriptionaland translational regulation. Bbk protein genomic DNA can be extractedand purified from human tissue by means well known in the art (forexample, see Berger, S. L., et al., Eds., Guide to Molecular CloningTechniques, Academic Press (1987)).

Alternatively mRNA can be isolated from any cell which produces orexpresses the protein, and used to produce cDNA by means well known inthe art (for example, see Berger, S. L., et al., Eds., Guide toMolecular Cloning Techniques, Academic Press (1987)). Preferably, themRNA preparation used will be enriched in mRNA coding for such Bbkprotein, either naturally, by isolation from cells which are producinglarge amounts of the protein, or in vitro, by techniques commonly usedto enrich mRNA preparations of specific sequences, including for examplesucrose gradient centrifugation, or PCR. cDNA can then be prepared forexample, by reverse transcription. The cDNA can then be amplified by PCRusing suitable primers.

For cloning into a vector, such suitable DNA preparations (either humangenomic DNA or cDNA) are randomly sheared or enzymatically cleaved,respectively, and ligated into appropriate vectors to form a recombinantgene (either genomic or cDNA) library. A DNA sequence encoding the Bbkprotein or its functional equivalents may be inserted into a DNA vectorin accordance with conventional techniques, including blunt-ending orstaggered-ending termini for ligation, restriction enzyme digestion toprovide appropriate termini, filling in of cohesive ends as appropriate,alkaline phosphatase treatment to avoid undesirable joining, andligation with appropriate ligases. Techniques for such manipulations aredisclosed, for example, by Sambrook, J., et al., Molecular Cloning: ALaboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press,Planview, N.Y. (1989), and are well known in the art.

Libraries containing the Bbk protein clones may be screened and a Bbkclone identified by any means which specifically selects for Bbk proteinDNA such as, for example, (a) by hybridization with an appropriatenucleic acid probe(s) containing a sequence specific for the DNA of thisprotein, or (b) by hybridization-selected translational analysis inwhich native mRNA which hybridizes to the clone in question istranslated in vitro and the translation products are furthercharacterized, or, (c) if the cloned genetic sequences are themselvescapable of expressing mRNA, by immunoprecipitation of a translated Bbkor fragment product produced by the host containing the clone.

Oligonucleotide probes specific for the protein which can be used toidentify clones to this protein can be designed from knowledge of theamino acid sequence of the Bbk protein. The sequence of amino acidresidues in a peptide is designated herein either through the use oftheir commonly employed three-letter designations or by theirsingle-letter designations. A listing of these three-letter andone-letter designations may be tound in textbooks such as Biochemistry,2ed., Lehninger, A., Worth Publishers, New York, N.Y. (1975). When theamino acid sequence is listed horizontally, the amino terminus isintended to be on the left end whereas the caiboxy terminus is intendedto be at the right end. The residues of amino acids in a peptide may beseparated by hyphens. Such hyphens are intended solely to facilitate thepresentation of a sequence.

Because the genetic code is degenerate, more than one codon may be usedto encode a particular amino acid (Watson, J. D., In: Molecular Biologyof the Gene, 3rd Ed., W. A. Benjamin, Inc., Menlo Park, Calif. (1977),pp. 356-357). The peptide fragments are analyzed to identify sequencesof amino acids which may be encoded by oligonucleotides having thelowest degree of degeneracy. This is preferably accomplished byidentifying sequences that contain amino acids which are encoded by onlya single codon.

Although occasionally an amino acid sequence may be encoded by only asingle oligonucleotide sequence, frequently the amino acid sequence maybe encoded by any of a set of similar oligonucleotides. Importantly,whereas all of the members of this set contain oligonucleotide sequenceswhich are capable of encoding the same peptide fragment and, thus,potentially contain the same oligonucleotide sequence as the gene whichencodes the peptide fragment, only one member of the set contains thenucleotide sequence that is identical to the exon coding sequence of thegene. Because this member is present within the set, and is capable ofhybridizing to DNA even in the presence of the other members of the set,it is possible to employ the unfractionated set of oligonucleotides inthe same manner in which one would employ a single oligonucleotide toclone the gene that encodes the peptide.

Using the genetic code (Watson, J. D., In: Molecular Biology of theGene, 3rd Ed., W. A. Benjamin, Inc., Menlo Park, Calif. (1977)), one ormore different oligonucleotides can be identified from the amino acidsequence, each of which would he capable of encoding the present Bbk orfragment protein. The probability that a particular oligonucleotidewill, in fact, constitute the actual protein coding sequence can beestimated by considering abnormal base pairing relationships and thefrequency with which a particular codon is actually used (to encode aparticular amino acid) in eukaryotic cells. Such "codon usage rules" aredisclosed by Lathe, et al., J. Molec. Biol. 183:1-12 (1985). Using the"codon usage rules" of Lathe, a single oligonucleotide sequence, or aset of oligonucleotide sequences, that contains a theoretical "mostprobable" nucleotide sequence capable of encoding the Bbk proteinsequences is identified.

The suitable oligonucleotide, or set of oligonucleotides, which iscapable of encoding a fragment of the Bbk protein gene (or which iscomplementary to such an oligonucleotide, or set of oligonucleotides)may be synthesized by means well known in the art (see, for example, S.A. Narang, Ed., Synthesis and Application of DNA and RNA, AcademicPress, San Diego, Calif.) and employed as a probe to identify andisolate the cloned Bbk protein gene by techniques known in the art.Techniques of nucleic acid hybridization and clone identification aredisclosed by Maniatis, et al., Eds., Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y.,(1982); Berger, et al., Eds., Guide to Molecular Cloning Techniques,Academic Press, San Diego, Calif., (1988); Sambrook, J., et al.,Molecular Cloning: A Laboratory Manual, 2d Ed., Cold Spring HarborLaboratory Press, Planview, N.Y. (1989); and by Hames, et al., Eds.,Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington,D.C., (1985), which references are herein incorporated by reference.Those members of the above-described gene library which are found to becapable of such hybridization are then analyzed to determine the extentand nature of the Bbk protein encoding sequences which they contain.

To facilitate the detection of the desired Bbk or fragment protein DNAencoding sequence, the above-described DNA probe is labeled with adetectable group or label. Such detectable group or label can be anymaterial having a detectable physical or chemical property. Suchmaterials have been well-developed in the field of nucleic acidhybridization and in general most any label useful in such methods canbe applied to the present invention. Particularly useful are radioactivelabels, such as ³² P, ³ H, ¹⁴ C, ³⁵ S, ¹²⁵ I or the like. Anyradioactive label may be employed which provides for an adequate signaland has a sufficient half-life. The oligonucleotide may be radioactivelylabeled, for example, by "nick-translation" by well-known means, asdescribed in, for example, Rigby, et al., J. Mol Biol. 113:237 (1977)and by T4 DNA polymerase replacement synthesis as described in, forexample, Deen, et al., Anal. Biochem. 135:456 (1983).

Alternatively, polynucleotides are also useful as nucleic acidhybridization probes when labeled with a non-radioactive marker such asbiotin, an enzyme or a fluorescent or chemiluminescent group. See, forexample, Leary, et al., Proc. Natl. Acad. Sci, USA 80:4045 (1983); Renz,et al., Nucl. Acids Res. 12:3435 (1984); and Renz, M., EMBO J. 6:817(1983).

Thus, the actual identification of the Bbk protein sequences permits theidentification of a theoretical "most probable" DNA sequence, or a setof such sequences, capable of encoding such a peptide. By constructingan oligonucleotide complementary to this theoretical sequence (or byconstructing a set of oligonucleotides complementary to the set of "mostprobable" oligonucleotides), one obtains a DNA molecule (or set of DNAmolecules), capable of functioning as a probe(s) for the identificationand isolation of clones containing the Bbk protein gene.

In an alternative way of cloning the Bbk protein gene, a library isprepared using an expression vector, by cloning DNA or, more preferably,cDNA prepared from a cell capable of expressing the Bbk protein, into anexpression vector. The library is then screened for members whichexpress the Bbk protein, for example, by screening, the library withantibodies to the Bbk protein.

The above discussed methods arc, therefore, capable of identifyinggenetic sequences which are capable of encoding Bbk proteins orfragments thereof. In order to further characterize such geneticsequences, and, in order to produce the recombinant protein, it isdesirable to express the proteins which these sequences encode. Suchexpression identities those clones which express proteins possessingcharacteristics of the Bbk proteins. Such characteristics may includethe ability to specifically bind antibody to the Bbk protein and theability to elicit the production of an antibody or antibodies which arecapable of binding to the Bbk protein.

To express the Bbk protein or a functional equivalent, or mutantthereof, transcriptional and translational signals recognizable by anappropriate host are necessary. The cloned Bbk encoding sequences,obtained, for example, through the methods described above, andpreferably in a double-stranded form, may be operably linked tosequences controlling transcriptional expression in an expressionvector, and introduced into a host cell, either prokaryotic oreukaryotic, to produce recombinant Bbk protein or a functionalequivalent thereof. Depending upon which strand of the Bbk encodingsequence is operably linked to the sequences controlling transcriptionalexpression, it is also possible to express Bbk antisense RNA or afunctional equivalent thereof.

Expression of Bbk in different hosts may result in differentpost-translational modifications which may alter the properties of theBbk. The present invention encompasses the expression of the Bbkprotein, or functional equivalent thereof, or Bbk mutant, in prokaryoticor eukaryotic cells, and particularly, eukaryotic expression ispreferred.

Preferred prokaryotic hosts include bacteria such as E. coli, Bacillus,Streptomyces, Pseudomonas, Salmonella, Serratia, etc. The most preferredprokaryotic host is E. coli. Other enterobacteria such as Salmonellatyphimurium or Serratia marcescens, and various Pseudomonas species mayalso he utilized. Under such conditions, the protein may not beglycosylated. The prokaryotic host must be compatible with the repliconand control sequences in the expression plasmid.

To express the Bbk protein (or a functional equivalent thereof) or Bbkmutant in a prokaryotic cell (such as, for example, E. coli, B.subtilis, Pseudomonas, Streptomices, etc.), it is necessary to operablylink the Bbk encoding sequence to a functional prokaryotic promoter.Such promoters may be either constitutive or, more preferably,regulatable (i.e., inducible or derepressible). Examples of constitutivepromoters include the int promoter of bacteriophage lambda, the blapromoter of the β-lactamase gene of pBR322, and the CAT promoter of thechloramphenicol acetyl transferase gene of pBR325, etc. Examples ofinducible prokaryotic promoters include the major right and leftpromoters of bacteriophage lambda (P_(L) and P_(R)), the trp, recA,lacZ, lacI, and gal promoters of E. coli, the α-amylase (Ulmanen, I., etal., J. Bacterial. 162:176-182 (1985)) and the sigma-28-specificpromoters of B. subtilis (Gilman, M. Z., et al., Gene 32:11-20 (1984)),the promoters of the bacteriophages of Bacillus (Gryczan, T. J., TheMolecular Bioloty of the Bacilli, Academic Press, Inc., N.Y. (1982)),and Streptomyces promoters (Ward, J. M., et al., Mol. Gen. Genet.203:468-478 (1986)). Prokaryotic promoters are reviewed by Glick, B. R.,(J. Ind. Microbial. 1:277-282 (1987)); Cenatiempo, Y. (Biochimie68:505-516 (1986)); and Gottesman, S. (Ann. Rev. Genet. 18:415-442(1984)).

Proper expression in a prokaryotic cell also requires the presence of aribosome binding site upstream of the gene-encoding sequence. Suchribosome binding sites are disclosed, for example, by Gold, L., et al.(Ann. Rev. Microbial. 35:365-404 (1981)).

Especially preferred eukaryotic hosts include mammalian cells either invivo, in animals or in tissue culture. General principles of mammaliancell culture are known in the art and are described, for example, inButler, M. and Dawson, M., Eds., Cell Culture LabFax, Bios ScientificPublishers Ltd., Oxford, UK and Academic Press, Inc., San Diego, Calif.,Publishers (1992), and references cited therein.

Expression of the Bbk in eukaryotic hosts requires the use of regulatoryregions functional in such hosts, and preferably eukaryotic regulatorysystems. A wide variety of transcriptional and translational regulatorysequences can be employed, depending upon the nature of the eukaryotichost. The transcriptional and translational regulatory signals can alsobe derived from the genomic sequences of viruses which intect eukaryoticcells, such as adenovirus, bovine papilloma virus, Simian virus, herpesvirus, or the like. Preferably, these regulatory signals are associatedwith a particular gene which is capable of a high level of expression inthe host cell.

In eukaryotes, where transcription is not linked to translation, suchcontrol regions may or may not provide an initiator methionine (AUG)codon, depending on whether the cloned sequence contains such amethionine. Such regions will, in general, include a promoter regionsufficient to direct the initiation of RNA synthesis in the host cell.Promoters from heterologous mammalian genes which encode mRNA productcapable of translation are preferred, and especially, strong promoterssuch as the promoter for actin, collagen, myosin, etc., can be employedprovided they also function as promoters in the host cell. Preferredeukaryotic promoters include the promoter of the mouse metallothionein Igene (Hamer, et al., J. Mol. Appl. Gen. 1:273-288 (1982)); the TKpromoter of Herpes virus (McKnight, S., Cell 31:355-365 (1982)); theSV40 early promoter (Benoist, et al., Nature (London) 290:304-310(1981)); and the HCMV promoter (Boshart, et al., Cell 41:521 (1985)); inyeast, the yeast gal4 gene promoter (Johnston, et al., Proc. Natl. Acad.Sci. USA 79:6971-6975 (1982); Silver, et al., Proc. Natl. Acad. Sci. USA81:5951-5955 (1984)) or a glycolytic gene promoter may be used.

As is widely known, translation of eukaiyotic mRNA is initiated at thecodon which encodes the first methionine. For this reason, it ispreferable to ensure that the linkage between a eukaryotic promoter anda DNA sequence which encodes the Bbk protein, or a functional equivalentthereof does not contain any intervening codons which are capable ofencoding a methionine. The presence of such codons results either in theformation of a fusion protein (if the AUG codon is in the same readingframe as the Bbk encoding DNA sequence) or a frame-shift mutation (ifthe AUG codon is not in the same reading frame as the Bbk encodingsequence).

If desired, a fusion product of the Bbk may be constructed. For example,the sequence coding for the Bbk or fragment thereof may be liked to asignal sequence which will allow secretion of the protein from or thecompartmentalization of the protein in, a particular host. Such signalsequences may be designed with or without specific protease sites suchthat the signal peptide sequence is amenable to subsequent removal.

Transcriptional initiation regulatory signals can be selected whichallow for repression or activation, so that expression of the operablylinked genes can he modulated. Of interest are regulatory signals whichare temperature-sensitive, such that by varying the temperature,expression can be repressed or initiated, or which are subject tochemical regulation, e.g., by a metabolite. Also of interest areconstructs wherein the Bbk mRNA and antisense RNA are provided in atranscribable form, but with different promoters or othertranscriptional regulatory elements such that induction of Bbk mRNAexpression is accompanied by repression of antisense RNA expression,and/or repression of Bbk mRNA expression is accompanied by induction ofantisense RNA expression.

Translational signals are not necessary when it is desired to expressBbk antisense RNA sequences.

If desired, the non-transcribed and/or non-translated regions 3' to thesequence coding for the Bbk protein can be obtained by theabove-described cloning methods. The 3'-non-transcribed region may beretained for its transcriptional termination regulatory sequenceelements; the 3'-non-translated region may be retained for itstranslation termination regulatory sequence elements, or for thoseelements which direct polyadenylation in eukaryotic cells. Where thenative expression control sequence signals do not functionsatisfactorily in the host cell, then sequences functional in the hostcell may be substituted.

The vectors of the invention may further comprise other operably linkedregulatory elements such as enhancer sequences, or DNA elements whichconfer tissue or cell-type specific expression on an operably linkedgene.

To transform a mammalian cell with the DNA constructs of the inventionmany vector systems are available, depending upon whether it is desiredto insert the Bbk DNA construct into the host cell chromosomal DNA, orto allow it to exist in an extrachromosomal form.

If the bbk DNA encoding sequence and an operably linked promoter areintroduced into a recipient eukaryotic cell as a non- replicating DNA(or RNA) molecule, which may either be a linear molecule or a closedcovalent circular molecule which is incapable of autonomous replication,then the expression of the Bbk protein may occur through the transientexpression of the introduced sequence.

Genetically stable transformants may be constructed with vector systems,or transformation systems, whereby bak DNA is integrated into the hostchromosome. Such integration may occur de novo within the cell or, in apreferred embodiment, be assisted by transformation with a vector whichfunctionally inserts itself into the host chromosome, for example, withretroviral vectors, transposons or other DNA elements which promoteintegration of DNA sequences into chromosomes. A vector is employedwhich is capable of integrating the desired gene sequences into amammalian host cell chromosome.

Cells which have stably integrated the introduced DNA into theirchromosomes are selected by also introducing one or more markers whichallow for selection of host cells which contain the expression vector inthe chromosome, for example, the marker may provide biocide resistance,e.g., resistance to antibiotics, or heavy metals, such as copper, or thelike. The selectable marker gene can either be directly linked to theDNA gene sequences to be expressed, or introduced into the same cell byco-transfection.

In another embodiment, the introduced sequence is incorporated into aplasmid or viral vector capable of autonomous replication in therecipient host. Any of a wide variety of vectors may be employed forthis purpose, as outlined below.

Factors of importance in selecting a particular plasmid or viral vectorinclude: the case with which recipient cells that contain the vector maybe recognized and selected from those recipient cells which do notcontain the vector; the number of copies of the vector which are desiredin a particular host; and whether it is desirable to be able to"shuttle" the vector between host cells of different species.

Preferred eukaryotic plasmids include those derived from the bovinepapilloma virus, vaccinia virus, SV40, and, in yeast, plasmidscontaining the 2-micron circle, etc., or their derivatives. Suchplasmids are well known in the art (Botstein, et al., Miami Wntr. Symp.19:265-274 (1982); Broach, J. R., The Molecular Biology of the YeastSaccharomyces: Life Cycle and Inheritance, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., pp. 445-470 (1981); Broach, J. R.,Cell 28:203-204 (1982); Bollon, et sl., J. Clin, Hematol. Oncol.10:39-48 (1980); Maniatis, T., "Gene Expression," In: Cell Biology: AComprehensive Treatise, Vol. 3, Academic Press, New York, pp. 563-608(1980)), and are commercially available. For example, mammalianexpression vector systems which utilize the MSV-LTR promoter to driveexpression of the cloned gene, and in which it is possible tocontransfect with a helper virus to amplify plasmid copy number, andintegrate the plasmid into the chromosomes of host cells, have beendescribed (Perkins, et (al., Mol. Cell Biol. 3:1123 (1983); Clontech,Palo Alto, Calif.).

Once the vector or DNA sequence containing the construct(s) is preparedfor expression, the DNA construct(s) is introduced into an appropriatehost cell by any of a variety of suitable means, including transfection.After the introduction of the vector, recipient cells are grown in aselective medium, which selects for the growth of vector-containingcells. Expression of the cloned gene sequence(s) results in theproduction of the Bbk protein, or in the production of a fragment ofthis protein. This expression can take place in a continuous manner inthe transformed cells, or in a controlled manner, for example,expression which follows induction of differentiation of the transformedcells (for example, by administration of bromodeoxyuracil toneuroblastoma cells or the like).

The expressed protein is isolated and purified in accordance withconventional conditions, such as extraction, precipitation,chromatography, affinity chromatography, electrophoresis, or the like.

Bbk can he purified by growing the transformed host cells under suitableconditions which are well known in the art, the cells can be harvestedand disrupted to extract total cellular protein. The protein can then,for example, be placed on a sizing column such as sepharose or agarosebeads, and proteins of the correct molecular weight can be collected.The predicted molecular weight of Bbk is 26.7 kD and it runs with anapparent molecular weight of approximately 37 kD on SDS polyacrylamidegels.

Further purification can be eftected by use of an anti-Bbk antibody.Such an antibody can be used to immunoprecipitate Bbk proteins from theset of cellular proteins of the correct approximate molecular weight.Such antibodies can, for example, be raised against polypeptidessynthesized according to the sequence or subsequences of the sequenceshown in FIG. 2. Alternatively, the antibodies can be raised againstfusion proteins, which contain Bbk sequences as well as those of otherproteins. After immunoprecipitation, the Bbk proteins can be releasedfrom the antibodies to provide a substantially pure preparation of Bbkprotein.

The bbk DNA coding sequences, of the present invention may be used toobtain Bbk antisense RNA genetic sequences, inasmuch as the antisenseRNA sequence will be that sequence found on the opposite strand of thestrand transcribing the peptide core's mRNA. The antisense DNA strandmay also be operably linked to a promoter in an expression vector suchthat transformation with this vector results in a host capable ofexpression of a Bbk antisense RNA in the transformed cell. Antisense RNAand its expression may be used to interact with an endogenous bbk DNA orRNA in a manner which inhibits or represses transcription or translationof the bbk genes in a highly specific manner. Use of antisense RNAprobes to block gene expression is described, for example, inLichtenstein, C., Nature 333:801-802 (1988).

Identification, Characterization and Use of Bbk Fragment CompositionsComprising the Bbk BH3 Domain

A novel domain within the Bbk molecule that appears to be both necessaryand sufficient for the known biological activities of Bbk has beenidentified. This domain, designated herein as the "Bbk BH3 domain," issufficient to mediate cell killing function and may be sufficient forphysical interaction with Bak. Mutation of Bbk BH3 domain sequences hasbeen demonstrated to reduce the apoptotic activity of the Bbk protein inRat1 fibroblast cells. These experiments demonstrate that the Bbk BH3domain is required for the cell killing and Bak binding activities ofthe Bbk protein. These observations suggest that Bbk modulates orregulates apoptosis through a mechanism that involves the Bbk BH3domain. As those of skill familiar with the present invention willappreciate, sequences comprising the Bbk BH3 domain are useful inmodulating apoptosis in cells. Similarly, compounds and compositionswhich are capable ol binding to the Bbk BH3 domain are useful as agentsfor the modulation of apoptotic activity in cells.

As used herein, the term "Bbk BH3 domain" refers to a protein domainfirst identified in Bbk, demonstrated herein to be essential for theinteraction of Bbk with Bak and for Bbk's cell killing function, and topeptides and/or molecules capable of mimicking its structure and/orfunction. In a preferred embodiment, the present invention comprises apeptide having the following amino acid sequence:

LRRLVALLEEEAE SEQ ID NO:1!

corresponding to amino acid residues 125-137 of Bbk, as well asfunctional equivalents thereof. By "functional equivalent" is meant apeptide possessing a biological activity or immunological characteristicsubstantially similar to that of the Bbk BH3 domain, and is intended toinclude "fragments", "variants", "analogs", "homologs", or "chemicalderivatives" possessing such activity or characteristic. Functionalequivalents of the Bbk BH3 domain, then, may not share an identicalamino acid sequence, and conservative or non-conservative amino acidsubstitutions of conventional or unconventional amino acids arepossible.

Reference herein to "conservative" amino acid substitution is intendedto mean the interchangeability of amino acid residues having similarside chains. For example, glycine, alanine, valine, leucine andisoleucine make up a group of amino acids having aliphatic side chains;serine and threonine are amino acids having aliphatic-hydroxyl sidechains; asparagine and glutamine are amino acids having amide-containingside chains; phenylalanine, tyrosine and tryptophan are amino acidshaving aromatic side chains; lysine, arginine and histidine are aminoacids having basic side chains; aspartic acid and glutamic acid areamino acids having acidic side chains; and cysteine and methionine areamino acids having sulfur-containing side chains. Interchanging oneamino acid from a given group with another amino acid from that samegroup would be considered a conservative substitution. Preferredconservative substitution groups include asparagine-glutamine,alanine-valine, lysine-arginine, phenylalanine-tyrosine andvaline-leucine-isoleucine.

In additional embodiments of the invention, there are provided peptideshaving the following amino acid sequence:

LRRLAALLEEEAE SEQ ID NO:2!

LRRLVALAEEEAE SEQ ID NO:3!

LRRLVALLEAAAE SEQ ID NO:4!

corresponding to alanine point mutants as shown in FIG. 9, which alsodemonstrate significant Bbk cell killing function. The Bbk BH3 domaindisclosed herein is uniquely involved in both cell killing and Bakbinding activity of Bbk.

The functional importance of the Bbk BH3 domain is likely to be relatedto its ability to mediate one or more protein/protein interactions withother Bcl-2 family members, or with other as yet unidentified cellularprotein(s). The present inventor does not intend to be bound by aparticular theory; however, regardless of its mechanism(s) of action,the Bbk BH3 domain in Bbk is of central importance for mediating theseprotein/protein interactions.

Agents capable of modulating Bbk BH3 domain mediated protein/proteininteractions may include peptides comprising the Bbk BH3 domain, as wellas mutants of the Bbk BH3 domain or of proteins comprising the Bbk BH3domain. A "mutant" as used herein refers to a peptide having an aminoacid sequence which dilters from that of the naturally occurring peptideor protein by at least one amino acid. Mutants may have the samebiological and immunological activity as the naturally occurring Bbk BH3domain peptide or the naturally occurring protein. However, thebiological or immunological activity of mutants may differ or belacking. For example, a Bbk BH3 domain mutant may lack the biologicalactivity which characterizes naturally occurring Bbk BH3 domain peptide,but may be useful as an antigen for raising antibodies against the BbkBH3 domain or for the detection or purification of antibodies againstthe Bbk BH3 domain, or as an agonist (competitive or non-competitive),antagonist, or partial agonist of the function of the naturallyoccurring Bbk BH3 domain peptide.

Modulation of Bbk BH3 domain mediated protein/protein interactions maybe effected by agonists or antagonists of Bbk BH3 domain peptides aswell. Screening of peptide libraries, compound libraries and otherinformation banks to identify agonists or antagonists of the function ofproteins comprising the Bbk BH3 domain is accomplished with assays fordetecting the ability of potential agonists or antagonists to inhibit oraugment Bbk BH3 domain binding, e.g., Bbk BH3 domain homodimerization orheterodimerization.

For example, high through-put screening assays may be used to identifycompounds that modulate the protein binding function of the Bbk BH3domain. Such screening assays facilitate the identification of compoundsthat accelerate or inhibit apoptosis by influencing protein/proteininteractions mediated by the Bbk BH3 domain. For example, an in vitroscreen for compounds that disrupt the Bbk BH3 domain interaction withBak comprises multiwell plates coated with Bak which are incubated witha labeled Bbk BH3 domain peptide probe in the presence of one or morecompounds to be tested. Molecules that specifically disrupt theinteraction could, in principle, bind to either the Bbk BH3 domain"ligand" or to the "receptor" domain in Bak. Either class of compoundwould be a candidate apoptosis-modulating agent.

Thus, the invention provides a method of screening for an agent capableof modulating apoptosis which comprises coating a multiwell plate withBak and incubating the coated multiwell plate with a labeled Bbk BH3domain peptide probe in the presence of an agent which it is desired totest, wherein disruption of Bbk BH3 domain interaction with Bakindicates that said agent is capable of modulating apoptosis. Agentsidentified by this method are also contemplated embodiments of theinvention.

Suitable labels include a detectable label such as an enzyme,radioactive isotope, fluorescent compound, chemiluminescent compound, orbioluminescent compound. Those of ordinary skill in the art will know ofother suitable labels or will he able to ascertain such using routineexperimentation. Furthermore, the binding of these labels to thepeptides is accomplished using standard techniques known in the art.

A high speed screen for agents that bind directly to the Bbk BH3 domainmay employ immobilized or "tagged" combinatorial libraries. Agents thatbind specifically to such libraries are candidates to be tested fortheir capacity to block Bbk/Bak interactions. As discussed above, suchagents may function as suppressors of apoptosis by either directlyinhibiting Bbk (and/or Bbk/Bak) function, or by increasing the effectiveactivity of endogenous Bcl-2 (or other Bcl-2 family member). Such agentswould be useful for suppressing aberrant apoptosis in degenerativedisorders or following ischemic injury.

Antibodies against the Bbk BH3 domain peptides of the invention may beused to screen cDNA expression libraries for identifying clonescontaining cDNA inserts encoding structurally related,immunocrossreactive proteins which may be members of the Bbk BH3 domainfamily of proteins. Screening of cDNA and mRNA expression libraries isknown in the art. Similarly, antibodies against Bbk BH3 domain peptidesare used to identify or purify immunocrossreactive proteins related tothis domain, or to detect or determnine the amount of proteinscontaining the Bbk BH3 domain in a cell or cell population, for example,in tissue or cells, such as lymphocytes, obtained from a patient. Knownmethods for such measurements include immunoprecipitation of cellextracts folllowed by PAGE, in sits detection by immunohistochemicalmethods, and ELISA methods, all of which are well known in the art.

Modulation of apoptosis according to the invention includes methodsemploying specific antisense polynucleotides complimentary to all orpart of the nucleotide sequences encoding proteins comprising the BbkBH3 domain disclosed herein. Such complimentary antisensepolynucleotides may include nucleotide additions, deletions,substitutions and transpositions, providing that specific hybridizationto the target sequence persists. Soluble antisense RNA or DNAoligonucleotides which can hybridize specifically to mRNA speciesencoding proteins comprising the Bbk BH3 domain, and which preventtranscription of the mRNA species and/or translation of the encodedpolypeptidc are contemplated as complimentary antisense polynucleotidesaccording to the invention. Production of proteins comprising the BbkBH3 domain is inhibited by antisense polynucleotides according to theinvention, and such antisense polynucleotides may inhibit apoptosis,senescence and the like, tnd/or reverse the transformed phenotype ofcells. A heterologous expression cassette maybe used to produceantisense polynucleotides in a transfectant or transgenic cell.Antisense polynucleotides also may be administered as solubleoligonucleotides to the external environment of the target cell, such asthe culture medium of cells in vitro or the interstitial fluid (e.g.,via the circulatory system) in vivo. Antisense polynucleotides and theiruse are known to those of skill, and are described, for example, inMelton, D. A., Ed, Antisense RNA and DNA, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1988).

The predicted biological activity of agents identified according to theinvention varies depending on the assumptions made regarding themechanism of Bbk/Bak function. For example, an agent which binds tightlyto the Bbk BH3 domain would be predicted to inhibit Bbk (and perhapsBbk/Bak) function. Assuming Bbk (and/or Bbk/Bak) is the active celldeath regulatory molecule, an agent that binds tightly to the Bbk BH3domain may inhibit Bbk function. Such agents might, therefore, exhibitanti-apoptotic activity under conditions in which Bbk has a demonstratedapoptotic effect. Agents in this class could have utility in treatingdiseases characterized by excessive or inappropriate cell death,including, for example, neuro-degenerative diseases and injury resultingfrom ischemia.

Peptidomimetics of Bbk BH3 domain peptide are also provided by thepresent invention, and can act as drugs for the modulation of apoptosisby, for example, blocking the function of proteins comprising the BbkBH3 domain or interfering with Bbk BH3 domain mediated interations.Peptidomimetics are commonly understood in the pharmaceutical industryto include non-peptide drugs having properties analogous to those ofthose of the mimicked peptide. The principles and practices ofpeptidomimetic design are known in the art and are described, forexample, in Fauchere J., Adv. Drug Res. 15: 29 (1986); and Evans et al.,J. Med. Chem. 30:1229 (1987). Peptidomimetics which bear structuralsimilarity to therapeutically useful peptides may be used to produce anequivalent therapeutic or prophylactic effect. Typically, suchpeptidomimetics have one or more peptide linkages optionally replaced bya linkage which may convert desirable properties such as resistance tochemical breakdown in vivo. Such linkages may include --CH₂ NH--, --CH₂S--, --CH₂ --CH₂ --, --CH═CH--, --COCH₂ --, --CH(OH)CH₂ --, and --CH₂SO--. Peptidomimetics may exhibit enhanced pharmacological properties(biological half life, absorption rates, etc.), different specificity,increased stability, production economies, lessened antigenicity and thelike which makes their use as therapeutics particularly desirable.

Immunization of animals with peptides comprising the Bbk BH3 domainalone or in conjunction with adjuvants by known methods can produceantibodies specific for the Bbk BH3 domain peptide. Antiserum obtainedby conventional procedures may be utilized for this purpose. Forexample, a mammal, such as a rabbit, may be immunized with a peptidecomprising the Bbk BH3 domain, thereby inducing the formation ofpolyclonal antibodies thereagainst. Monoclonal antibodies also may begenerated using known procedures. Such antibodies can be used accordingto the invention to detect the presence and amount of peptidescomprising the Bbk BH3 domain.

The Bbk BH3 domain peptides of the invention may be used for thedetection of Bbk and other proteins by means of standard assaysincluding radioimmunoassays and enzyme immunoassays.

It will be appreciated by those of skill that the precise chemicalstructure of peptides comprising the Bbk BH3 domain will vary dependingupon a number of factors. For example, a given protein may be obtainedas an acidic or basic salt, or in neutral form, since ionizable carboxyland amino groups are found in the molecule. For the purposes of theinvention, then, any form of the peptides comprising the Bbk BH3 domainwhich retains the therapeutic or diagnostic activity of the naturallyoccurring peptide is intended to be within the scope of the presentinvention.

The term "substantially homologous" as used herein refers to the abilityof a first DNA sequence encoding Bbk to hybridize to a second DNAsequence encoding the foregoing, under stringent conditions, forexample, at about 0.1x sodium citrate sodium chloride buffer (SSC) at atemperature of about 65° C. The term "substantially pure" means that theprotein or molecule of interest is essentially free from any otherdetectable biological constituents. A "fragment" of a molecule such asBbk is meant to refer to any variant of the molecule which possess thebiological activity of the Bbk protein. A "variant" of a molecule ismeant to refer to a molecule substantially similar in structure andbiological activity or immunological characteristics to either theentire molecule, or to a fragment thereof. Thus, provided that twomolecules possess a similar activity, they are considered variants asthat term is used herein even if the composition or secondary, tertiary,or quaternary structure of one of the molecules is not identical to thatfound in the other, or it the sequence of amino acid residues is notidentical. An "analog" of a molecule is meant to refer to a moleculesubstantially similar in function to either the entire molecule or to afragment thereof. As used herein, a molecule is said to be a "chemicalderivative" of another molecule when it contains additional chemicalmoieties not normally a part of the molecule. Such moieties may improvethe molecule's solubility, absorption, biological half life, etc. Themoieties nmay alternatively decrease the toxicity of the molecule,eliminate or attenuate any undesirable side effect of the molecule, etc.Moieties capable of mediating such effects are described, for example,in Remington's Pharmaceutical Sciences (1980). Procedures for couplingsuch moieties to a molecule are well known in the art. By the term"modulate" is intended, for the purposes of the present invention, theinduction of apoptosis by the administration of the Bbk protein of theinvention, an active fragment thereof, a functional equivalent thereof,and/or the suppression or induction of apoptosis by the administrationof a Bbk hybrid or Bbk mutant, or the administration of a vectorcontaining cDNA encoding any of the foregoing, to the particular cellsof an individual suffering from any degenerative disorder which resultsin inappropriate cell growth, for example, including lymphomas,genotypic tumors, cancer, or, disorders characterized by inappropriatecell death, for example, including AIDS which results in T-cell death,in order to stabilize inappropriate cell proliferation or inappropriatecell death and preferably to restore normal cell behavior.

By the term "administration" is intended any mode of administrationwhich results in the delivery of the therapeutic agent across the cellmembrane and into the desired cell. The site of administration and cellswill be selected by one of ordinary skill in the art based upon anunderstanding of the particular degenerative disorder being treated. Inaddition, the dosage, dosage frequency, and length of course oftreatment, can be determined and optimized by one of ordinary skill inthe art depending upon the particular degenerative disorder beingtreated. The particular mode of administration can also be readilyselected by one of ordinary skill in the art and can include, forexample, oral, intravenous, subcutaneous, intramuscular, etc., with therequirement that the therapeutic agent cross the cell membrane. Thetherapeutic agent of the present invention can be the Bbk protein and/orfunctional equivalents thereof and/or Bbk hybrids or Bbk mutants and/ora vector containing CDNA encoding the foregoing. By the term"therapeutic agent" is intended the present Bbk protein, fragments,functional equivalents and/or hybrids or mutants thereof as well asvectors containing DNA encoding any of the foregoing. The presenttherapeutic agent can be administered alone or in combination withand/or concurrently with other suitable drugs and/or courses of therapy.By the term "degenerative disorder" is intended for purposes of thisinvention, any disorder characterized by inappropriate cellproliferation or inappropriate cell death or in some cases, both. By theterm "inappropriate cell proliferation" is intended a statisticallysignificant increase in cell number as compared to the proliferation ofthat particular cell type in the normal population. Also included aredisorders whereby a cell is present and/or persists in an inappropriatelocation, e.g., the presence of fibroblasts in lung tissue after acutelung injury. For example, such cells include cancer cells which exhibitthe properties of invasion and metastasis and are highly anaplastic.Such cells include but are not limited to, cancer cells including, forexample, tumor cells. By the term "inappropriate cell death" is intendeda statistically significant decrease in cell number as compared to thepresence of that particular cell type in the normal population. Suchunderrepresentation may be due to a particular degenerative disorder,including, for example, AIDS (HIV), which results in the inappropriatedeath of T-cells, autoimmune diseases which are characterized byinappropriate cell death. By the term "autoimmune disease" is intended adisorder caused by an immune response directed against self antigens.Such diseases are characterized by the presence of circulatingautoantibodies or cell-mediated immunity against autoantigens inconjunctions with inflammatory lesions caused by immunologicallycompetent cells or immune complexes in tissues containing theautoantigens. Such diseases include systemic lupus erythematosus (SLE),rheumatoid arthritis.

By the term "suppression" is intended for the purposes of this inventionthe result achieved by administering an amount of a therapeutic agentcontaining Bbk hybrids or Bbk mutants thereof effective to suppressapoptosis in an individual suffering from a degenerative disordercharacterized by inappropriate cell death. Suppression of apoptosis isachieved when the numbers of the particular affected cell type remainstable or increase in number to a level within the range observed in thenormal cell population. By the term "stable" is intended the stateachieved when a statistically significant decrease in cell number is nolonger observed in the individual being treated, as compared to the cellnumber observed at the onset of the course of treatment. By the term"induction" is intended for the purposes of this invention the resultachieved by the administration of an amount of a therapeutic agentcontaining the Bbk of the invention effective to induce apoptosis incells of an individual ,suffering from a degenerative disordercharacterized by inappropriate cell proliferation. The induction ofapoptosis is achieved when cell numbers remain stable or decrease to alevel within the range observed in the normal cell population. One ofordinary skill in the art can readily determine whether the induction ofapoptosis has been achieved.

By the term "Bbk hybrid" is intended for the purposes of this invention,proteins which are hybrid proteins of the present Bbk proteins,fragments thereof, and/or functional equivalents or mutants thereof,with other apoptosis associated proteins encoded by genes including, forexample, Bcl-2, Bax, c-myc, LMW5-HL, Bbk, Bcl-X_(L), Bcl-X_(S), BHRF-1,Mcl-1, A1 and ced9, fragments thereof and/or functional equivalentsthereof, in order to produce a protein which exhibits enhanced,decreased, or intermediate apoptosis induction or suppression activityas compared to the activity of Bbk alone. Such hybrids can be produced,for example, by fusing the first half of the coding region of the bbkcDNA with the second half of the coding region of the cDNA for bcl-2, orbax, or bcl-x_(L), or bcl-x_(S) or vice versa. Additionally, by addingor replacing segments of bcl-2, bax, bcl-x_(L) or bcl-x_(S) to the bbkcDNA, chimeric gene products of therapeutic value can be generated. Oneof ordinary skill in the art can readily produce and employ such hybridsusing techniques well known in the art. One of ordinary skill in the artcan readily determine whether a particular hybrid exhibits enhanced,decreased or intermediate apoptosis induction or suppression activityusing known screening methods and as described herein. By the term"normal cell behavior" is intended for the purposes of this invention,cells in which apoptosis proceeds normally. Normal cell behavior isobserved in an organism which is able to remove senescent, damaged, orabnormal cells that could interfere with organ function or develop intotumors. Apoptosis which proceeds normally represents a coordinatedcellular response to noxious stimuli that are not immediately lethal.

By the term "patient" or "individual" is intended for the purposes ofthe present invention, animals, including humans and mammals, who sufferfrom a degenerative disorder. By the term "Bbk mutant" is intended forthe purposes of the present invention a mutant of Bbk which exhibits thereverse (apoptosis suppression) activity of the Bbk protein of theinvention due to the substitution of one or more amino acids orcorresponding nucleotides. By the term "apoptosis associated proteinBbk" is intended for the purposes of the present invention both theisolated naturally occurring and isolated recombinantly produced protein(i.e., synthetic Bbk) which exhibits, inter alia, apoptosis inductionfrom human tissue including, for example, tumor cells and establishedhuman cell lines, and from tissues of other animals including mammals.This term includes any analog, homolog, mutant or derivative of isolatednaturally occurring Bbk including fragments having less than thenaturally occurring number of amino acids, such as partial fragments ofnatural or synthetic Bbk which retain the biological or immunologicalcharacteristics of the polypeptide disclosed in this application. Thisterm also includes any peptide which contains the sequence of anisolated naturally occurring Bbk protein, or analog or homolog thereof,together with one or more flanking amino acids, which retains thebiological or immunological characteristics of the Bbk protein of theinvention.

Construction and Identification of Antibodies Raised Against Bbk,Functional Equivalents, Fragments, Hybrids, or Mutants Thereof

In the following description, reference will be made to variousmethodologies well-known to those skilled in the art of immunology.Standard reference works setting forth the general principles ofimmunology include the work of Catty, D., Antibodies. A PracticalApproach, Vol. 1, IRL Press, Washington, D.C. (1988); Klein, J.,Immunology: The Science of Cell-Noncell Discrimination, John Wiley &Sons, New York (1982); Kennett, et al., Monoclonal Antibodies.Hybridoma: A New Dimension in Biological Analyses, Plenum Press, NewYork (1980); Campbell, A., "Monoclonal Antibody Technology," In: Burdon,R., et al., Eds., Laboratory Techniques in Biochemistry and MolecularBiology, Vol. 13, Elsevier, Amsterdam (1984); and Eisen, H. N., in:Davis, B. D., et al., Eds., Microbiology, 3d ed., Harper & Row,Philadelphia (1980).

An antibody is said to be "capable of binding" a molecule if it iscapable of specifically reacting with the molecule to thereby bind themolecule to the antibody. The term "epitope" is meant to refer to thatportion of a hapten which can be recognized and bound by an antibody. Anantigen may have one, or more than one epitope. An "antigen" is capableof inducing an animal to produce antibody capable of binding to anepitope of that antigen. The specific reaction referred to above ismeant to indicate that the antigen will react, in a highly selectivemanner, with its corresponding antibody and not with the multitude ofother antibodies which may be evoked by other antigens.

The term "antibody" (Ab) or "monoclonal antibody" (Mib) as used hereinis meant to include intact molecules as well as fragments thereof (suchas, for example, Fab and F(ab)₂ fragments) which are capable of bindingan antigen. Fab and F(ab)₂ fragments lack the Fc fragment of intactantibody, clear more rapidly from the circulation, and may have lessnon-specific tissue binding of an intact antibody (Wahl, et al., J. NucLMed. 24:16-325 (1983)).

The antibodies of the present invention have specificity to one or moreepitopes present on the Bbk peptide, or an idiotype on the present Bbk.The antibodies of the invention can be polyclonal or monoclonal,provided that they are made with the present Bbk polypeptide or fragmentthereof as the immunogen. Both of these types of antibodies can beutilized in the applications described herein.

The present antibodies can be used to detect the presence of the presentBbk protein in a human tissue sample. The present Bbk protein can bedetected by contacting the sample with an imaging-effective amount ofthe present detectably labeled appropriate antibody and detecting thelabel, thereby establishing the presence of the Bbk protein in thesample. Detection can be carried out by imaging in vivo. The Bbk proteincan also be detected by known immunoassay techniques, including, forexample, RIA, ELISA, etc., using appropriate antibodies according to theinvention.

The antibodies of the present invention are prepared by any of a varietyof known methods. For example, cells expressing the Bbk protein can beadministered to an animal in order to induce the production of serumcontaining polyclonal antibodies that are capable of binding the Bbkprotein. For example, the Bbk protein or fragment thereof is chemicallysynthesized and puritfed by HPLC to render it substantially tree ofcontaminants. Such a preparation is then introduced into an animal inorder to produce polyclonal antisera of high specific activity.

Polyclonal antibodies can be generated in any suitable animal including,for example, mice, rabbits or goats. The Bbk immunogenic peptide orfragment thereof can be injected by itself or linked to appropriateimmunoactivating carriers, such as Keyhole limpet hemocyanin (KLH). SeeCatty, D., Ed., Antibodies, A Practical Handbook, Vols. I and II, IRLPress, Washington, D.C. (1988).

Monoclonal antibodies can be prepared in various ways using techniqueswell understood by those having ordinary skill in the art. For example,monoclonal antibodies can be prepared using hybridoma technology(Kohler, et al., Nature 256:495 (1975); Kohler, et al., Eur. J. imninol.6:511 (1976); Kohler, et al., Eur. J. Immunol. 6:292 (1976); Hammerling,et al., In: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y.,pp. 563-681 (1981)); Roger H. Kennett, et al., Eds., MonoclonalAntibodies - Hybridomas: A New Dimension in Biological Analysis, PlenumPress (1980). In general, such procedures involve immunizing an animalwith the present Bbk protein, or a fragment thereof. The splenocytes ofsuch animals are extracted and fused with a suitable mycloma cell line.Any suitable mycloma cell line may be employed in accordance with thepresent invention. After fusion, the resulting hybridoma cells areselectively maintained in HAT medium, and then cloned by limitingdilution as described by Wands, et al., Gastroenterol. 80:225-232(1981). The hybridoma cells obtained through such a selection are thenassayed to identify clones which secrete antibodies capable of bindingthe Bbk protein.

Through application of the above-described methods, additional celllines capable of producing antibodies which recognize epitopes of thepresent Bbk protein can be obtained.

For example, additional hybridomas which produce monoclonal antibodieswhich enable the detection of the present Bbk protein can be easilyproduced and isolated with minimal screening. Hybridomas producingmonoclonal antibodies specific for epitopes which are found on thepresent Bbk protein are most effectively produced by first immunizing ananimal from which hybridomas can be produced such as, for example, aBalb/c mouse, with initial subcutaneous injections of Freund's adjuvant,followed by booster injections within a few days. The fusion can becarried out using any of the techniques commonly known to those ofordinary skill in the art. The screening of the hybridomas to determinewhich ones are producing monoclonal antibodies specific for the presentpeptide is straightforward and can be accomplished in a standard ELISAor RIA format. For example, in an RIA screening forrnat the culturesupernatant, or ascites fluid from a hybridoma producing monoclonalantibody is reacted with ¹²⁵ I-peptide. The isolation of otherhybridomas secreting mAbs of the same specificity as those describedherein can be accomplished by the technique of anti-idiotypic screening.Potocmjak, et al., Science 215:1637 (1982). Briefly, an anti-idiotypic(anti-Id) antibody is an antibody which recognizes unique determinantsgenerally associated with the antigen-binding site of an antibody. An Idantibody can be prepared by immunizing an animal of the same species andgenetic type (e.g., mouse strain) as the source of the mAb with the mAbraised against the present Bbk protein or fragment thereof to which ananti-Id is being prepared. The immunized animal will recognize andrespond to the idiotypic determinants of the immunizing antibody byproducing an antibody to these idiotypic determinants (the anti-Idantibody).

By using an anti-Id antibody which is specific for idiotypicdeterminants on a given mAb, it is then possible to identify other Bcell or hybridoma clones sharing that idiotype. Idiotypic identitybetween the antibody product of two clones makes it highly probable thatthe antibody products of the two clones recognize the same antigenicepitopes.

The anti-Id antibody may also be used as an "immunogen" to induce animmune response in yet another animal, producing a so-calledanti-anti-Id antibody. The anti-anti-Id may be epitopically identical tothe original mAb which induced the anti-Id.

Thus, by using antibodies to the idiotypic determinants of a mAb, it ispossible to identity other clones expressing antibodies of identicalspecificity.

Accordingly, mAbs generated against the present Bbk protein may be usedto induce anti-Id antibodies in suitable animals, such as BALB/c mice.Spleen cells from such immunized mice are used to produce anti-Idhybridomas secreting anti-Id mAbs. Further, the anti-Id mAbs can becoupled to a carrier such as keyhole limpet hemocyanin (KLH) and used toimmunize additional BALB/c mice. Sera from these mice will containanti-anti-Id antibodies that have the binding properties of the originalmAb specific for the antigen epitope. The anti-Id mAbs thus have theirown idiotypic epitopes, or "idiotopes" structurally similar to theepitope being evaluated.

For replication, the hybridoma cells of this invention may be cultivatedin vitro or in vivo. Production of high titers of mAbs in vivoproduction makes this the presently preferred method of production.Briefly, cells from the individual hybridomas are injectedintraperitoneally into pristane-primed BALB/c mice to produce ascitesfluid containing high concentrations of the desired mAbs. MAbs ofisotype IgM or IgG may be purified from such ascites fluids, or fromculture supernatants, using column chromatography methods well known tothose of skill in the art.

Of special interest to the present invention are antibodies which areproduced in humans, or are "humanized" (i.e., non- immunogenic in ahuman) by recombinant or other technology such that they will not beantigenic in humans, or will be maintained in the circulating serum of arecipient for a longer period of time.

Humanized antibodies may be produced, for example by replacing animmunogenic portion of an antibody with a corresponding, but non-immunogenic portion (i.e., chimeric antibodies) (Robinson, et al.,International Patent Publication PCT/US86/02269; Akira, et al., EuropeanPatent Application 184,187; Taniguchi, M., European Patent Application171,496; Morrison, et a., European Patent Application 173,494;Neuberger, et al., PCT Application WO 86/01533, Cabilly, et al.,European Patent Application 125,023; Better, et al., Science240:1041-1043 (1988); Liu, et al., Proc. Natl. Acad. Sci. USA84:3439-3443 (1987); Liu, et al., J. Immbunol. 139:3521-3526 (1987);Sun, et al., Proc. Natl. Acad. Sci. USA 84:214-218 (1987); Nishimura, etal., Canc. Res. 47:999-1005 (1987); Wood, et al., Nature 314:446-449(1985)); Shaw, et al., J. Natl. Cancer Inst. 80:1553-1559 (1988).General reviews of "humanized" chimeric antibodies are provided byMorrison, S. L. (Science, 229:1202-1207 (1985)) and by Oi, et al.,BioTechniques 4:214 (1986)).

Suitable "humanized" antibodies can be alternatively produced asdescribed by Jones, et al., Nature 321:552-525 (1986); Verhocyan, etal., Science 234:1534 (1988), and Beidler, et al., J. Immninol.141:4053-4060 (1988).

The present Bbk protein, fragments thereof, hybrids thereof, Bbkmutants, or antibodies thereto can be utilized in immunoassays for thedetection of the Bbk protein in a human tissue sample. For example,antibodies against the present Bbk protein can be used to detect thepresent Bbk protein in a human tissue sample. The immunoassays can becompetitive or sandwich, as is otherwise well known and they all dependon the formation of antibody-antigen immune complex. These assays arewell known to those of skill in the art.

For purposes of the assays, the antibody or antigen can he immobilizedor labeled. There are many carriers to which the antibody/antigen can bebound for immobilization and which can be used in the present invention.Well-known carriers include but are not limited to glass, polystyrene,polypropylene, polyethylene, dextran, nylon, amylases, natural andmodified celluloses, polyacrylamides, agaroses, and magnetite. Thenature of the carrier can be either soluble to some extent or insolublefor purposes of the invention. Those skilled in the art will know manyother suitable carriers for binding the antibody or antigen, or will beable to ascertain such, using routine experimentation.

Depending on the particular embodiment of the invention, one or more ofthe antibodies or antigen(s) peptide(s) will be coupled with adetectable label such as an enzyme, radioactive isotope, fluorescentcompound, chemiluminescent compound, or bioluminescent compound.

Those of ordinary skill in the art will know of other suitable labelsfor binding to the antibodies or antigen(s) peptide(s) or will be ableto ascertain such using routine experimentation. Furthermnore, thebinding of these labels to the antibodies or antigen(s) can be doneusing standard techniques commonly known to those of ordinary skill inthe art.

The antibodies or antigen peptide(s) can be bound to an enzyme. Thisenzyme, in turn, when later exposed to its substrate will react with thesubstrate in such a manner as to produce a chemical moiety which can bedetected, as, for example, by spectrophotometric or fluorometric means.Examples of enzymes that can be used to detectably label are amylatedehydrogenase, staphylococcal nuclease, delta-5-steroidisomerase, yeastalcoholdehydrogenase, α-glycerophosphate dehydrogenase, triose phosphateisomerase, alkaline phosphatase, asparaginase, glucose oxidase,β-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphatedehydrogenase, glucoamylase, and acetylcholinesterase.

The presence of an antibody or antigen can also be detected by labelingthe antibody or antigen with a radioactive isotope. The presence of theradioactive isotope can be determined by such means as the use of agammacounter or a scintillation counter. Isotopes which are particularlyuseful are ³ H, ¹²⁵ I, ³² P, ³⁵ S, ¹⁴ C, ⁵¹ Cr, ³⁶ Cl, ⁵⁷ Co, ⁵⁹ Fe, ⁷⁵Se, and ¹⁵² Eu.

It is possible to detect the presence of the antibody or antigen bylabeling the antibody or antigen peptide with a fluorescent compound.When the fluorescently labeled antibody or antigen peptide is exposed tolight of the proper wavelength, its presence can then be detected due tofluorescence of the dye. Among the most common fluorescent labelingcompounds are fluorescein isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde, and fluorescamine.

Another way in which the antibody or antigen can be detectably labeledis by coupling it to a chemiluminescent compound. The presence of thechemiluminescent-tagged antibody or antigen peptide is then determinedby detecting the presence of luminescence that arises during the courseof a chemical reaction. Examples of particularly useful chemiluminescentlabeling compounds are luminol, isoluminol, aromatic-acridinium ester,imidaxole, acridinium salt, and oxalate ester.

Likewise, a bioluminescent compound may also be used to label theantibody or antigen peptide. Bioluminescence is a special type ofchemiluminescence which is found in biological systems and in which acatalytic protein increases the efficiency of the chemiluminescentreaction. The presence of a bioluminescent binding partner would bedetermined by detecting the presence of luminescence. Importantbioluminescent compounds for purposes of labeling are luciferin,luciferase, and aequorin.

The antibodies or antigen peptide(s) for use in the assay of theinvention are ideally suited for the preparation of a kit. Such a kitmay comprise a carrier means being compartmentalized to receive in closeconfinement one or more container means such as vials, tubes, and thelike, each of said container means comprising one of the separateelements to be used in the method.

For example, one of the container means may comprise a first antibodybound to an insoluble or partly soluble carrier. A second container maycomprise soluble, detectably-labeled second antibody, in lyophilizedform or in solution. The carrier means may also contain a thirdcontainer means comprising a detectably labeled third antibody inlyophilized form or in solution. Such a kit can be used for sandwichassays.

In addition, the carrier means may also contain a plurality ofcontainers each of which comprises difterent, predetermined amounts ofthe present Bbk peptide. These latter containers can then be used toprepare a standard curve into which can be used to interpolate theresultsobtained from the sample containing the unknown amount of thepresent Bbk protein.

Imaging can be carried out in vitro or in vivo. In vitro imaging can bedone with the labels mentioned previously. In vivo imaging is done withdiagnostically effective labeled antibodies. The term "diagnosticallyeffective" means that the amount of detectably labeled antibodyadministered is sufficient to enable detection of the site of Bbkprotein presence when compared to a background signal.

Generally, the dosage of detectably-labeled antibody or antigen(s) fordiagnosis will vary depending on considerations such as age, condition,sex, and extent of disease in the patient, counterindications, if any,and other variables, to be adjusted by the individual physician. Dosagecan very from 0.01 mg/kg to 2,000 mg/kg, preferably 0.1 mg/kg to 1,000mg/kg.

The term "diagnostically labeled" means that the antibody has attachedto it a diagnostically detectable label.

There are many different imaging labels and methods of labeling known tothose of ordinary skill in the art. Examples of the types of labelswhich can be used in the present invention include radioactive isotopesand paramagnetic isotopes.

For diagnostic in vivo imaging, the type of detection instrumentavailable is a major factor in selecting a given radionuclide. Theradionucleotide chosen must have a type of decay which is detectable fora given type of instrument. In general, any conventional method forvisualizing diagnostic imaging can be utilized in accordance with thisinvention.

Another important factor in selecting a radionuclide for in vivodiagnosis is that the half-life of a radionucleotide be long enough sothat it is still detectable at the time of maximum uptake by the target,but short enough so that deleterious radiation upon the host isminimized. Ideally, a radionuclide used for in vivo imaging will lack aparticulate emission, but produce a large number of photons in a 140-200ke V range, which may be readily detected by conventional gamma cameras.

For in vivo diagnosis, radionucleotides may be bound to antibody orantigen either directly or indirectly by using an intermediaryfunctional group. Intermediary functional groups which are often used tobind radioisotopes which exist as metallic ions to antibody or antigenare diethylenetriaminepentaacetic acid (DTPA) andethlenediaminetetracetic acid (EDTA). Typical examples of metallic ionswhich can be bound to immunoglobulin are ^(99m) Tc,¹²³ I, ¹¹¹ In, ¹³¹ I,⁹⁷ Ru, ⁶⁷ Cu, ⁶⁷ Ga, ⁷² As, ⁸⁹ Zr, and ²⁰¹ T1.

The antibodies used in the method of the invention can also be labeledwith paramagnetic isotopes for purposes of in vivo diagnosis. Elementswhich are particularly useful (as in magnetic resonance imaging (MRI)techniques) in this manner include ¹⁵⁷ Gd, ⁵⁵ Mn, ¹⁶² Dy, ⁵² Cr, and ⁵⁶Fe.

Preparations of the imaging antibodies for administration includesterile aqueous or non-aqueous solutions, suspensions, and emulsions.Examples of non-aqueous solvents are propyleneglycol,polyethyleneglycol, vegetable oil such as olive oil and injectableorganic esters such as ethyloleate. Aqueous carriers include water,alcoholic/aqueous solutions, emulsions or suspensions, including salineand buffered media, parenteral vehicles including sodium chloridesolution, Ringer's dextrose, dextrose and sodium chloride, lactatedRinger's or fixed oils. Intravenous vehicles include fluid and nutrientreplenishers, electrolyte replenishers, such as those based on Ringer'sdextrose, and the like. Preservatives and other additives may also bepresent, such as, for example, antimicrobials, anti-oxidants, chelatingagents, and inert gases and the like. See, generally, Remington'sPharmaceutical Science, 16th ed. Mac Eds. 1980.

Of course, the expressed Bbk protein is an intracellular protein.Accordingly, those of skill will recognize that in vivo diagnostic andtherapeutic methods employing the antibodies of the invention mayrequire some mechanism by which such antibodies can detect Bbk in thecell. One such method is to introduce the antibodies or fragmentsthereof into the cell itself across the cell membrane. This may beaccomplished, for example, by attaching the antibody to a ligand forwhich the target cell contains receptor sites. The antibody can thus betransported into the cell membrane or across the cell membrane alongwith the ligand. Suitable ligands include growth factors and cytokinesthat are internalized upon receptor binding. Suitable growth factorsinclude epidermal growth factor (EGF), tumor growth factor alpha(TGF-α), fibroblast growth factor (FGF), insulin, and insulin-likegrowth factors 1 and 2 (IGF- 1 and IGF-2). Suitable cytokines includeG-CSF, GM-CSF, erythrol)oietin, IL-1 and IL-2. It is noted that thereare also receptors that carry nutrients and vitamins into cells. Thesenutrients are suitable for use as ligands in the present invention andinclude folate, dihydrofolate, tetrahydrofolate and vitamin B12.

The choice of a carrier ligand will depend on several factors, as thoseof skill will appreciate. These include, for example, the kinetics ofthe ligand and its receptor, and of overall transport, which may includepassive or active, with actively transported ligands preferred. Themeans of attaching the antibody to the ligand also will vary withinlimits, and may be, for example, covalent or ionic, bearing in mind thatsuch attachment should not unacceptably alter ligand-receptor affinity.

Examples of receptors suitable for such applications include thereceptor for low density lipoprotein (LDL), which has been shown tocontain all the information necessary for receptor endocytosis, Davis etal., J. Cell Biol. 107(6/3): Abstr. No. 3112 (1988), as well as knownbrain-specific receptors such as those for dopamine. In this regard, itwill be appreciated that the ligand may itself be an antibody orfragment specific for the receptor, to which may be conjugated theantibody of the invention.

Moreover, those of skill may find it particularly desirable to employantibody fragments of the invention (such as, for example, Fab orF(ab')₂ fragments), which are less likely to interfere with theligand-receptor interaction, and may be more easily transported acrossthe cell membrane. Single-chain antibodies may prove preferable forthese and other reasons, as will be appreciated by those of skill.

When an antibody is to be transported into the cell's membrane or intothe cell as described above, it will be preferred to diagnostically ortherapeutically label the antibody in such a way that the label will berelatively more effective when the antibody is bound to its antigenicsite on the Bbk protein. This may be accomplished, for example, byemploying a label which becomes active or detectable as a result offormation of the antigen-aintibody complex. Alternatively, the antibodyitself may be labeled in such a way that antigen-antibody complexformation induces a conformational change in the antibody to expose ormore fully expose the previously unexposed or less fully exposed label.All of the above criteria, and others, will be apparent to those ofskill in carrying out these aspects of the invention.

It is also possible to utilize liposomes having the antibodies of thepresent invention in their membranes to specifically deliver theantibodies to the target area. These liposomes can be produced so thatthey contain, in addition to the antibody, such therapeutic agents asdrugs, radioisotopes, lectins and toxins, which would act at the targetsite.

Pharmaceutical Compositions

Pharmaceutical compositions containing a therapeutically effectiveamount of the present Bbk protein, functional equivalents, fragmentsand/or hybrids an/or mutants thereof, as well as vectors containing cDNAencoding one or more of the foregoing, are useful for treating patientssuffering from degenerative disorders characterized by inappropriatecell death or inappropriate cell proliferation.

Hybrids of Bbk include hybrids of Bbk and for example Bcl-2, ced-9,Bcl-X_(L), Bcl-X_(S), Bax, Mcl-1, c-myc, LMW5-HL, BHRF-1, Bak, Bik andA1. Such hybrids exhibit enhanced, decreased or intermediate apoptosisinduction or suppression activity as compared to the activity of Bbkalone. These hybrids can be readily selected, produced and employed byone or ordinary skill in the art. Pharmaceutical compositions accordingto the invention thus will contain a therapeutically effective amount ofthe present Bbk protein, functional equivalents, fragments and/orhybrids and/or mutants thereof, and may optionally contain one or morepharmaceutically acceptable carriers and/or excipients, known to thoseof ordinary skill in the art. Administration, dosage and frequency, andlength of the course of treatment can be readily optimized for aparticular patient by one of ordinary skill in the art For example, thepresent pharmaceutical composition can be formulated as sterile aqueousor non-aqueous suspensions or emulsions, as described above, for examplefor solutions for intravenous administration.

Therapeutic Applications

Programmed cell death is a process in which cells undergo nuclearcondensation and fragmentation during normal development of healthytissues and organs. The process is essential in maintaining the balancebetween growth of new cells and elimination of old cells. When apoptosisdoes not work properly, either by causing cells to die prematurely or bypreventing them from dying when scheduled, various disorders develop.

The present apoptosis associated Bbk protein, functional equivalents,fragments and/or hybrids and/or mutants thereof as well as vectorscontaining cDNA encoding the foregoing are useful for treatingdegenerative disorders, which disorders are characterized byinappropriate cell death or inappropriate cell proliferation. Particulardisorders may involve diflerent cell types whereby it may be desirableto induce apoptosis in one cell type while suppressing apoptosis in theother. For example, it may be desirable to suppress apoptosis in lungtissue cells in a patient suffering from acute lung injury byadministering the Bbk mutant protein of the invention (or by effectingexpression of such Bbk mutant protein in those cells) while inducingapoptosis in fibroblast cells which may be present in the lung due tothe inflammatory response by administering the Bbk protein of theinvention (or by effecting Bbk protein expression in those cells).

The therapeutic agents of the present invention can be administered asdiscussed above with the requirement that the agent must cross the cellmembrane. The therapeutic agent can be administered alone, incombination with or during the course of treatment with other acceptabletherapies known in the art for treating a particular disorder. Forexample, the present therapeutic agents can be administered to induceapoptosis in a cancer patient who is also undergoing classic cancertherapy including, for example, radiation therapy, chemotherapy, andtreatment with anti-cancer drugs including, for example, topoisomeraseinhibitors, alkylating agents, antimetabolites, and hormone antagonists.Further, the present therapeutic agents can also be administeredconcurrently with gene therapy. For example, the present therapeuticagents can be administered to a patient suffering from a degenerativedisorder of the central nervous system while the patient is concurrentlyundergoing gene therapy to replenish neutrophic hormones.

Premature widespread apoptosis (inappropriate cell death) causes much ofthe damage associated with degenerative disorders including, forexample, AIDs, chemotherapy and radiation, and tissue atrophy. In AIDspatients, lymphocytes are activated even in the asymptomatic phase ofthe HIV infection, and those cells die prematurely by apoptosis. Suchdisorders may admit of treatment by administration of a Bbk mutantprotein.

Those of skill will appreciate that administration of the variousproteins of the invention to particular target cells or tissues, asdescribed herein, is intended to comprehend the administration of theproteins themselves as well as the expression by the target cells ortissues of the nucleotide sequences encoding those proteins by variousknown means and in accordance with the teachings of the presentspecification.

Degenerative disorders characterized in inappropriate cell proliferationinclude cancer, autoimmune disorders, tissue hypertrophy, andinflammatory disorders including inflammation arising from acute tissueinjury including, for example, acute lung iniury. These disorders can betreated by administering the present Bbk protein or functionalequivalent.

Cancers arise when changes in DNA cause the anomalous accumulation ofcells. The comparative rates of cell division and cell deaths determinehow fast a cancer grows. Some cancer cells divide more slowly thannormal cells, but the cancer may still expand because of prolonged celllife span. Apoptosis is an efficient method for preventing malignanttransformation because it removes cells with genetic lesions. Defectiveapoptosis can promote cancer development, both by allowing accumulationof dividing cells and by obstructing removal of genetic variants withenhanced malignant potential. The present therapeutic agents, includingthe present Bbk protein, functional equivalents, fragments, and hybridsthereof, along with vectors containing cDNA encoding the one or more ofthe foregoing, can be administered to cancer patients to induceapoptosis.

Many types of cancer can be treated by the administration of the presenttherapeutic agents, including for example, carcinomas, sarcomas, andleukemia/lymphomas, including for example, carcinomas such asadenocarcinomas, squamous carcinomas, carcinoma of the organs includingbreast, colon, head, neck, etc.; sarcomas including chondrosarcoma,melanosarcoma, etc.; and leukemia and lymphomas including acutelymphomatic leukemia, acute myelogenous leukemia, non- Hodgkin'slymphoma, Burkitt's lymphoma, B-cell lymphomas, T-cell lymphomas, etc.Other conditions amenable to treatment using the present therapeuticagent include fungal infections.

The present therapeutic agents can be used to treat autoimmune diseases.Random gene recombination and somatic hypermutation can potentiallygenerate autoreactive T and B lymphocytes throughout life. Under normalconditions immature lymphocytes that bind autoantigens die by apoptosis.However, a defect in the deletion of these lymphocytes predisposes oneto autoimmunity.

The present therapeutic agents can be administered to patients sufferingfrom autoimmune disorders to induce apoptosis in autoreactive Tlymphocytes, for example, in patients suffering systemic lupuserythematosus. Other autoimmune diseases amenable to treatment bysuppressing or inducing apoptosis through the administration of thepresent therapeutic agents include, for example, rheumatoid arthritis,myasthenia gravis, Grave's disease, Hashimoto's thyroiditis,insulin-resistent diabetes, allergic rhinitis, asthma, functionalautonomic abnormalities, juvenile insulin-dependent diabetes, Addison'sdisease, idiopathic hypoparathyroidism, spontaneous infertility,premature ovarian failure, pemphigus, Bullous pemphigoid, primarybiliary cirrhosis, autoimmune hemolytic anemia, idiopathicthrombocytopenic purpura, idiopathic neutropenia, Goodpasture'ssyndrome, rheumatoid arthritis and Sjogren's syndrome.

The present therapeutic agents can be used to treat inflammationresulting from acute lung injury, by inducing apoptosis. The diseaseprocess begins with an explosive inflammatory response in the alveolarwall. In the aftermath of the resulting tissue destruction, extensivefibroproliferation of the alveolar air space ensues, consisting offibroblasts, capillaries and their connective tissue products. Fukuda,Y., et al., Am. J. Pathol. 126:171-182 (1987). An important mechanismfor the systematic elimination of the foregoing is apoptosis, i.e.,programed cell death.

The present therapeutic agents can also be used to treat degenerativedisorders due to premature or excessive cell loss during aging which canlead to organ disfunction and disease. Such degenerative disordersinclude degenerative diseases of the central nervous system due to agingor other tactors which result in the death of neurons. The presenttherapeutic agents containing Bbk mutant protein or hybrids thereof canbe administered to a patient suffering from such a degenerative disorderto suppress apoptosis. Further, the present therapeutic agents can beadministered concurrently with gene therapy to provide genes encodingneutrophic hormones including, for example, nerve growth factor. Otherconditions amenable to treatment utilizing the present therapeuticagents include, for example, Alzhcimer's disease.

One of ordinary skill in the art can readily identify other degenerativedisorders characterized by inappropriate cell death or inappropriatecell proliferation or both which are amenable to treatment using thepresent therapeutic agents. The present therapeutic agents can includethe Bbk protein itself, as well as fragments, functional equivalentsand/or hybrids and/or mutants thereof, which are administered to atarget cell. Alternatively, therapeutic agents according to theinvention can be administered by infecting the target cell with a vectorcontaining cDNA encoding one or more of the foregoing. The presenttherapeutic agents can be administered to the desired target cell asdiscussed below, for example, by choosing a receptor on the target cellsurface which is specific for that cell type. The present therapeuticagents can be administered alone or in combination with other acceptabledrug therapies. Further, the present therapeutic agents can beadministered concurrently with other acceptable therapies specific forthe particular degenerative disorder being treated. For example, thepresent therapeutic agents can be administered concurrently withchiemotherapeutic agents, gene therapy, or the like. Whether it is theBbk protein itself or the a vector encoding the protein, the therapeuticagent must cross the cell membrane.

One method for introducing the Bbk protein or fragments thereof into thecell's membrane or into the cell itself is by attaching the protein to aligand for which the target cell contains receptor sites. The proteincan thus be transported into the cell membrane or across the cellmembrane along with the ligand.

The choice of a carrier ligand will depend on several factors, asdiscussed herein and known to those of skill. Suitable tissue- specificreceptors include: Brain: nerve growth factor receptor (NGF-R); breast:prolactin receptor; stomach: gastrin receptor; skin: melanocytestimulating hormone receptor (MSH-R), liver: asialoglycoproteinreceptor; thyroid: thyroid stimulating hon-one receptor (TSH-R);ovaries: luteinizing hormonereceptor (LH-R), testis: human chorionicgonadotrophin receptor (hCG-R), T-cells: T-cell receptors; B cells:CD19; lung hyaluronate receptor CD44 isoform 4V (J. Cell. Biol. 124,7182, 1994). In this regard, it will be appreciated that the ligand maybe an antibody or fragment specific for the receptor, to which may beconjugated the Bbk protein of the invention.

It may be desirable to employ active Bbk fragments according to theinvention which are less likely to interfere with the ligand- receptorinteraction, and which may be more easily transported across the cellmembrane.

When a protein is to be transported across the cell's membrane or intothe cell as described above and the ligand is an antibody, it will bepreferred to diagnostically or therapeutically label the protein in sucha way that the label will be relatively more effective when the proteinis bound, such as, for example, by means analogous to those describedherein in the context of antibody transport.

It is also possible to utilize liposomes having the proteins of thepresent invention in their membrane to specifically deliver the presentBbk proteins to the target area. These liposomes can be produced so thatthey contain, in addition to the Bbk protein, such other therapeuticagents including drugs, radioisotopes, lectins and toxins, which wouldbe released at the target site.

A preferred manner for administering the Bbk encoding nucleotidesequences (and their functional equivalents and/or hybrids and/ormutants) for diagnostic or therapeutic purposes is by the use of viralvectors. Suitable viral vectors for gene transfer include retroviruses(reviewed in Miller, et al., Methods Enzyrmol. 217:581-599 (1993))including human immunodeficiency virus (HIV), adenovirus derivatives(for exanmples see Erzurum, et al., Nucleic Acids Res. 21:1607-12(1993); Zabner, et al., Nat. Genet. 6:75-83 (1994); Davidson, et al.,Nat. Genet. 3:219-223 (1993)), adeno-associated virus (AAV), (i.e., seeFlotte, et al., Proc. Natl. Acad. Sci. USA 90:10613-7 (1993)) and Herpesvirus sectors (i.e., see Anderson, et al., Cell. Mol Neurobiol.13:503-15 (1993)). Other suitable viruses can be readily selected andemployed by those of ordinary skill in the art. Other methods for DNAdelivery include liposome mediated gene transfer (Alton, et al., Nat.Genet. 5:135-42 (1993); Nabel, et al., Proc. Natl. Acad. Sci. USA90:11307-11 (1993)).

The use of viral vectors for introduction of genes into mammalian cellsis also reviewed, for example, in Varmus, Science 240(4858):1427 (1988);Eglitis et al., BioTechniques 6,7:608 (1988); Jaenisch, Science240(4858):1468 (1988); and Bernstein et al., Genet. Eng. (N.Y) 7:235(1985).

For the purposes of the present invention, it may be preferred to employan attenuated viral or retroviral strain. Thus, for example, it ispossible to use as vectors for the DNA sequences of the inventionretroviruses having attenuated cytopathicity, such as HIV- 2_(ST) (Konget al., Science 240(4858):1525 (1988)) or HIV-2_(UC1) (Evans et al.,Science 240(4858):1523 (1988)), which enter neural cells by aCD4-dependent mechanism (Funke et al., J. Exp. Med. 165:1230 (1987)).The neurobiology of HIV infections is described, for example, in Johnsonet al., FASEB J. 2(14):2970 (1988). Those of skill will be able totarget different cell populations having known susceptibilities toviruses by the exercise of routine skill. For example, CD4 is known tohave a variant transcript in the human brain, with its highest contentin forebrain (Maddon et al., Cell 47:333 (1986). Possible methods totarget retroviral gene expression to specific cell types are reviewed byBoris-Lawrie and ft. Temin Curr. Opin. Genet. Dev. vol. 3, p.102-9(1993).

Ideally, then, the choice of a gene delivery system will be made bythose of skill, keeping in mind the objectives of efficient and stablegene transfer, with an appropriate level of gene expression, in atissue-appropriate manner, and without any adverse effects. See, forexample, Wolff et al., Rheum. Dis. Clin. North Am. 14(2):459 (1988).With respect to delivery to a central nervous system target, many viralvectors, including HIV, offer the advantage of being able to cross theblood-brain barrier (Johnson et al., FASEB J. 2(14):2970 (1988)).

Diagnostic Applications

Antibodies raised against the present Bbk protein, fragments, functionalequivalents, or hybrids or mutants thereof can be used to detect the Bbkprotein in a human tissue sample, as well as to diagnose degenerativedisorders associated with the expression of the Bbk protein. Further,such antibodies can also be used to monitor the progress of degenerativedisorders associated with the expression of the Bbk protein.

Any source of human cells is suitable for use in the diagnostic testingin the present invention. The cells can be isolated from any humantissue including for example, heart, lung, tumor cells, brain, placenta,liver, skeletal muscle, kidney and pancreas. Extraction ot proteins fromthe cell sample may be performed by any of the many means known in theart. For example, cells may be lysed by a detergent by mechanical means.if desired, nucleic acids can be removed from the cell preparation byenzymatic digestion or by precipitation. Such means are well known inthe art.

Antibodies can be generated which are immunoreactive with the Bbkproteins by the methods set forth herein. Appropriate antibodies canthen be screened using the natural gene products of bbk.

The extracted proteins from the cell sample may be contacted with theantibody under suitable conditions for antibody-antigen complexformation. Generally, such conditions are physiological conditions. Theprotein extract may be bound to a solid support such a nitrocellularfilter or a microtiter plate.

The antibody will generally hear a label which is a radio label, aflorescent label, or an enzyme conjugate which under appropriateconditions produces, for example, a colored reaction product. Antibodiesand antibody labeling are described herein and known to those of skill.Alternatively, if the antibody is not labeled, it can be detected bymeans of a second antibody from another species which is reacted withthe first antibody. Suitable assay techniques, labels and means ofdetection are discussed herein.

A parallel sample to the test sample is employed to provide the control.The control sample consists of an equivalent amount of proteinsextracted from cells, preferably in the same manner as those of the testsample. The amount of protein can readily be determined by employingtechniques well known in the art, including, for exaniple, the Lowry orBradford techniques. The cells used for preparing the control sample maybe selected from cells of the same cell type as the test cells, isolatedfrom a normal human not suffering from the degenerative disorder fromwhich the human from which the test sample was taken suffers, cells ofthe same cell type as the test sample isolated from an establishednormal cell line, and cells from the human who is being tested, whichcell type is different from the cell type of the test cells.

Test samples can also be screened for elevated levels of mRNAtranscribed from the bbk gene, according to methods well known in theart. For example, RNA extracted from B-cells may be used, oralternatively mRNA may be isolated from total cellular RNA. The mRNA maybe purified, for example, by affinity chromatography on oligo (dTcellulose) which binds to the poly (A) tract at the 3' end of most mRNA.As is well known to those skilled in the art, it is essential thatribonuclease activity be minimized during preparation and assaying.

A DNA probe may be selected from any of the protein coding sequences ofthe bbk gene. Preferably, the probe will be selected from sequences ofthe 5' or 1st exon of the gene so that RNA can be detected. Preferably,the probe contains at least 15 nucleotides of the bbk sequence. In orderto perform the hybridization, it is desirable that the probe be singlestranded. Thus, if the probe is double stranded, it should be denaturedto a single stranded form. Means for denaturing are well known in theart, including alkali or heat treatment. The probe can then be contactedwith the RNA derived from the cell sample under conditions wherehomologous RNA-DNA hybrids form and are stable. Such conditions are wellknown in the art. Means for detecting hybrids are many and well known,but often involve the use of radiolabeled probes and nucleases whichdegrade single stranded DNA. Other methods known in the art may be used.

Control samples can be derived from any of these cell sources describedabove for use in the antibody diagnostic tests. Samples and controlsshould preferably be prepared in parallel under similar conditions.

The diagnostic methods and compositions of the present invention areuseful for determining whether a disease/degenerative disorder is linkedto abnormal Bbk expression, to the expression of Bbk mutants, as well asfor determining the effect of over expression or loss of expression ofBbk in animal models such as transgenic mice and/or homozygous nullmice. Methods for determining whether a disease/degenerative disorder islinked to abnormal Bbk expression include analyzing Bbk expression indiseased tissue as compared to normal tissue by for example, Northernand/or Western blots, as well as by other assay methods readily chosenand employed by those of ordinary skill in the art. Once It has beendetermined that a disease/degenerative disorder is linked to abnormalBbk expression, the disease/disorder can be diagnosed in an individual.

The following examples are offered by way of illustration, not by way oflimitation.

EXAMPLES

A. Materials and Methods

1. Yeast two-hybrid analysis. Components of the yeast two-hybrid systemwere obtained from Clontech Laboratories (Catalog numbers K1605-1,K1605-D, HL4006AE). These included the GAL4 binding domain fusion vectorpAS2, yeast strains Y190 and Y187, and human lymphocyte cDNA activationdomain library. Bak was subcloned into the pAS2 vector using standardprotocols (Sambrook, J., et al., Molecular Cloning: A Laboratory Manual,2d Ed., Cold Spring Harbor Laboratory Press, Planview, N.Y. (1989)). Tocreate an in-frame fusion with Bak the pAS2 vector was modified bydigesting with NdeI, making the ends blunt with Klenow fragment of DNApolymerasc I, and re-ligating. The Bak gene was removed from pcDNA1/amp(described in co-pending U.S. application Ser. No. 08/321,071, filed 11Oct. 1994, which is a continuation-in-part of U.S. application Ser. No.08/287,427, filed 9 Aug. 1994 (bak is referred to therein as bcl-y)) bydigestion with BamHI and EcoRI, made blunt with the Klenow fragment ofDNA polymerase I, and ligated into the modified pAS2 vector that hadbeen digested with SmaI and treated with calf intestinal phosphatase toremove terminal phosphates. The Bak/GAL4 binding domain fusion vector(pAS2/BakΔC) was DNA sequenced across the fusion point to verify thatthe GAL4 reading frame had been preserved through the cloning junctionand into the Bak open reading frame. Two-hybrid analysis,β-galactosidase filter assays, and detection of false positives wereperformed using the Bak/GAL4 binding domain bait and lymphocyte cDNAGAL4 activation domain library following the manufacturer'sinstructions. Plasmid DNAs from the positive clones were isolated andtransformed into E. coli as described by the manufacturer. Bacterialclones were analyzed further by restriction enzyme analysis and DNAsequencing.

2. Additional Plasmid Constructs. All plasmid constructs were made usingstandard protocols (Samnbrook, J., et al., Molecular Cloning: ALaboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press,Planview, N.Y. (1989)). A modified foim of the mammalian expressionplasmid pcDNA3 (Clontech) was created in which the influenzahemagglutanin (HA) epitope tag (MGYPYDVPDYASLS) (SEQ ID NO:29) had beeninserted between the HindIII and XhoI sites of the polylinker cloningregion to generate pcDNA3/HA. The Bbk gene obtained in the two-hybridscreen (pACT/Bbk) was removed from the GAL4 activation domain plasmid onan XhoI fragment and subcloned into the XhoI site of the pcDNA3/HAvector described above to create pcDNA3/HABbk. This results in anin-frame fusion ol Bbk with the HA tag with the HA tag at the N-terminusof Bbk.

The deletion mutant Δ1-105 was obtained directly from the two-hybridscreen and subcloned in a manner simlar to full length Bbk to createpcDNA3/HAΔ1-105. The mutant pcDNA3/HAΔ142-249 was created by digestingpcDNA3/HABbk with PflMI and XbaI to remove sequences between nucleotides338-958 and subsequently replacing the deleted sequences with apolymerase chain reaction (PCR) generated PflMI to XbaI DNA fragmentcorresponding to Bbk nucleotides 338-476. An in-frame stop codon wasincorporated after amino acid 141.

The alanine point mutants pcDNA/HAPM-LVLEE (L₁₂₅ V₁₂₉ L₁₃₂ E₁₃₄ E₁₃₅replaced with A residues), pcDNA/HAPM-V (V₁₂₉, replaced with A residue),pcDNA/HAPM-L (L,₁₃₂ replaced with A residue), pcDNA/HAPM-EE (E₁₃₄ E₁₃₅replaced with A residues) were created by replacing wild type Bbksequences between nucleotides 338-476 (a PflMI fragment) withPCR-generated PflMI fragments that have incorporated alanine codons atthe designated positions. Bbk genes carrying the alanine point mutationswere removed from the pcDNA/HA vectors and also cloned into the Xholpolylinker site of pACT to create pACT/PM-LVLEE, pACT/PM-V, pACT/PM-L,and pACT/PM-EE. These plasmids generate an in-frame fusion between theBbk mutants and the Gal4 activation domain for use in yeast two-hybridanalysis.

Plasmid pRcCMV/HABbkBH3 expressing Bbk amino acid residues 117-166,which encompass the Bbk BH3 domain, was constructed as described below.A fragment of the Bbk gene encoding amino acid residues 117-166 wasgenerated by PCR (incorporating a stop codon after amino acid 166) andsubsequently cloned into the XhoI site of pcDNA3/HA (see above) that hadbeen made blunt with the Klenow fragment of DNA polymerase. The HAtag/Bbk BH3 fusion gene was then removed on an HindIII/XbaI fragment andsubcloned into the HindII/XbaI sites of pRcCMV (Clontech). This cloningresults in the in-frame fusion of the HA tag and the Bbk BH3 domain foruse in mammalian cell transfections. A control plasmid encoding wildtype Bbk was similarly constructed by removing the HindIII/XbaI fragmentfrom pcDNA3/HABbk (see above) and subcloning into the HindII/XbaI sitesof pRcCMV.

A glutathione S-transferase fusion of Bbk (GST-Bbk) was created bysubcloning a BglII to EcoRi fragment of Bbk into the BamHI to EcoRIsites of the plasmid pGEX2TK (Pharmacia). The BglII to EcoRI fragment ofBbk was created in several steps. First the initiating methionine of Bbkwas removed by replacing a BglII to Bsu36I fragment of pcDNA3/HABbk witha double-stranded oligonuleotide adapter. The Xbal site of this modifiedpcDNA3/HABbk plasmid was then converted to an EcoRI site using EcoRIlinkers.

Other plasmids expressing Bak, Bik, Bax, Bcl-x_(L), Bcl-2 and EpsteinBarr virus BHRF1 have been previously described (Boyd , J. M., et al.,Oncogene 11:1921 (1995); Chittenden ,T. EMBO J. 14(22):5589 (1995);co-pending U.S. application Ser. No. 08/321,071, filed 11 Oct., 1994,which is a continuation-in-part of U.S. application Ser. No. 08/287,427,filed 9 Aug., 1994 (bak is referred to therein as bcl-y)).

3. Northern Blot Analysis. Human fetal and adult multiple tissueNorthern blots were purchased from Clontech Laboratories and hybridizedsequentially to ³² P-labeled probes encompassing the entire codingregions of Bbk and β-actin following the supplier's protocol.

4. In vitro translation. ³⁵ S-methionine labeled proteins weresynthesized in vitro using the TnT T7/T3 coupled reticulocyte lysatesystem (Promega), following the manufacturer's procedures. Translationproducts were subjected to SDS polyacrylamide electrophoresis. The gelwas fixed, incubated in a flourography enhancing solution (Amplify,Amersham), dried, and subjected to autoradiography at -70° C.

5. Western blot analysis. COS7 cells were cultured in DMEM supplementedwith 10%, fetal calf serum and L-glutamine. Cells were transfected with2 μg of plasmid DNA using LipofectAMINE (Gibco/BRL), and cell lysateswere prepared 24 hours after transfection. Portions of the cells'extracts (approximately 100 mg) were electrophoresed on an SDSpolyacrylamide gel, and transferred to a nylon membrane by standardmethods (Harlow, E., et al., Antibodies: A Laboratory Manual (1988)).The blot was incubated with the anti-HA epitope monoclonal antibody12CA5 (Klodziej, P. A., et al., Meth. Enzymol. 194:508-519 (1991)),which was subsequently detected with a secondary antibody using the ECLsystem (Amersham).

6. Transient transfection analysis. Rat1, HeLa, and BT549 cells werecultured at 37° C., 7% CO₂ in DMEM with 10% fetal calf serum, 4 nML-glutamine, 50 units/ml penicillin, and 50 μg/ml streptomycin. Cellswere plated at 3.5=10⁴ cells/well in 24 well tissue culture dishes 24hours before transfection. A plasmid encoding E. coli β-galctosidase(pRcCMV/βgal, 0.16 μg) was mixed with a total of 0.42 μg plasmid(s) ofinterest as defined in the figure legends. The plasmid mixture was addedto 25 μl OPTIMEM (Gibco/BRL) and subsequently mixed with 27 μlLipofectAMINE solution (2 μl stock solution diluted with 25 μl OPTIMEM).After a 30 minute incubation at room temperature the plasmid mixtureswere diluted with 200 μl OPTIMEM and added to cells that had been rinsedonce with OPTIMEM. After 4 hours at normal growth conditions the cellswere fed with 250 μl DMEM, 20% fetal calf serum, 4 mM L-glutamine andthen allowed to grow for 24 hours under normal conditions. Cells werethen washed with phosphate buffered saline (PBS), fixed with 2%gluteraldehyde, 2% parafoimaldehyde, 49.2 mM sodium phosphate (pH 7.3)for 5 minutes at 4° C. and washed twice with PBS.β-galactosidase-expressing cells are identified as blue cells after a1-4 hour incubation with 80 mM Na₂ HPO₄, 20 mM NaH₂ PO₄, 1.3 mM MgCl₂, 1mg/ml X-Gal (diluted from a 20 mg/ml stock solution prepared indimethylformamide), 3 mM K₃ Fe(CN)₆, 3 mM K₄ Fe(CN)₆ /3H₂ O. Live bluecells are identified as those that retain a flat morphology while deadblue cells are round.

7. In vitro protein interactions. Glutathione S-transferase fusionprotein of Bbk (GST-Bbk) was expressed in E. coli from plasmidpGEX2TK-Bbk and purified by affinity chromatography using glutathioneagarose (Smith, D. B. and Johnson, K. S., Gene 67:31-40 (1988)). ³⁵S-methionine labeled HA-epitope tagged Bak, Bax, Bik, and Flag-epitope(Kodak) tagged Bcl-x_(L) was expressed iin vitro using a coupledtranscription/translation system in rabbit reticulocyte lysates(Promega). Labeled proteins were precleared with 10% glutathione-agarose in 10 mM HEPES buffer (pH 7.2) containing 0.25% NP-40, 142.5 mMKCl, 5 mM MgCl₂, 1 mM EGTA (Buffer A). GST-Bbk was added (finalconcentration 1-3 μM) and the mixtures incubated for 60 minutes at 4° C.Protein complexes were captured with 10% glutathione-agarose and washedtwice with buffer A and once with buffer A without NP-40. Proteins wereeluted from the beads by incubation in SDS-PAGE sample bufler at 100° C.for 5 minutes and loaded onto 4-20% SDS-polyacrylamide gels (Novex).Following electrophoresis, gels were fixed and incubated in aflourotgraphy enhancing solution (Amplify, Amersham). The gels weredried and subjected to autoradiography at -70° C.

8. Liquid culture β-galactosidase assays. The affinity of Bbk/Bak andmutant Bbk/Bak interactions was quantitated using a liquidβ-galactosidase assay with o-nitrophenylgalactoside (ONPG) as substrateas described by the manufacturer's protocol (Clontech). Plasmids usedfor analysis are as stated in the figure legends.

B. Results

1. Identification of Bak-interacting proteins by yeast two-hybridanalysis.

Bak is found expressed in many cells (co-pending U.S. application Ser.No. 08/321,071, filed 11 Oct., 1994, which is a continuation-in-part ofU.S. application Ser. No. 08/287,427, filed 9 Aug., 1994, abandoned (bakis referred to therein as bcl-y); Kiefer, M. C., et al., Nature 374:736(1995)). Overexpression of Bak induces death by apoptosis. It wasbelieved that regulators or effectors of apoptosis might bind to Bak.Proteins that interact with Bak were therefore identified using theyeast two-hybrid system (U.S. Pat. No. 5,283,173). The principle of thismethodology is summarized in FIG. 1.

GAL4 is a yeast transcriptional activator protein that has two distinctdomains, the DNA-binding domain and the transcription activation domain(FIG. 1A). The DNA-binding domain binds to a specific DNA sequenceelement in the GAL4 promoter, thus bringing the transcription activationdomain in proximity to the promoter where it functions to stimulatetranscription. It has been demonstrated that these domains areseparable, although when separated they cannot function to stimulatetranscription. Transcriptional activation can, however, be restored if alink between the two separated domains is made. Such a link can be madeby expressing the GAL4 domains as hybrid proteins where these domainsare fused to heterologous proteins (protein X and protein Y in FIGS.1B,C) that are known to interact. In the scenario shown in FIG. 1 theGAL4 binding domain serves to bring protein X to the GAL4 promoter andsubsequent interaction with protein Y in turn brings the GAL4 activationdomain to the promoter where it can stimulate transcription. The GAMApromoter, or a promoter containing the GAMA DNA sequence element, can beused to direct the transcription of selectable marker genes (such asHIS3) and reporter genes (such as lacZ).

The system described above can be used to study the interaction ofproteins that are known to interact as well as to identify and isolatenovel interacting proteins. In the latter case, a protein of interest isfused to the GAL4 binding domain and used as "bait" to captureinteracting proteins that are expressed as GAL4 activation domainfusions. In the experiments described here a GAL4 DNA-binding domain/Bakfusion protein was used as the bait to isolate Bak-interacting proteinsexpressed as GAL4 activation domain fusions generated from a cDNAlibrary of Epstein-Barr Virus-transformed human B lymphocytes(Clontech). Two-hybrid analysis, selection for interacting clones,β-galactosidase filter assays, and identification of false positiveswere performed following the manufacturer's specifications (Clontech).

2. Sequence analysis of an avid Bak-binding clone, bbk.

Eleven of the most avidly binding clones (as judged by the intensity ofblue color in β-galactosidase filter assays) were determined by DNAsequencing to be varying length clones of the same gene. Restrictionenzyme analysis was used to identify the approximate sizes of theseclones. Clone bbk was the largest of the clones and was therefore chosenfor exhaustive DNA sequencing of both the top and bottom strands. Thesequence of clone bbk is shown in FIG. 2. Six of the eleven clones havethe same sequence startpoint as bbk while there are five clones thathave deletions of varying length (up to nucleotides 33. 151, 178, 242,and 364 of clone bbk). Three of the eleven clones have an intact 3'termintis as determined by the presence of a polyadenylated (polyA)tail. The absence of a polyA tail in the remaining clones is likely tobe due to abbenant priming during cDNA synthesis due to the AT-richnature of the gene in this region. Nonetheless, the remaining cloneshave 3' termini that are within 30 bases of the true 3' terminus(including clone bbk, which is 10 bases short).

The sequence of bbk was compared to the Genbank database using the NCBIBLAST program. This analysis has identified numerous expressed sequencetag (EST) cDNAs (Accession Numbers: H26516, H42839, H59025, H59896,H59897, H72004, H72005, H89857, H90702, R02556, R02674, R07849, R07901,R36543, R38463, k58365, R78883, R78977, R85622) with significanthomology (Poisson Sum P(N) values less than or equal to 5.5e-26) to bbk.These cDNAs also begin and end within several bases of the endpoint ofbbk suggesting that bbk is a nearly full length clone.

Bbk encodes an open reading frame (ORF) of 249 amino acids beginningwith nucleotide number 51 and ending at nucleotide number 799. This ORFis in the same frame as that predicted by the GAL4 activation domainwhich is fused N-terminal to Bbk. The sequence surrounding the predictedinitiating methionine codon is consistent with the Kozak consensussequence (Kozak, M. Nucleic Acids Res. 15:8125 (1987)) and there are noadditional methionines or in-frame stop codons in the putativeuntranslated bbk sequence present between the Gal4 activation domain andthe Bbk ORF. Thus, it is believed that the true ORF of the Bbk gene hasbeen identified. It is interesting to note that several of the clonesisolated by two-hybrid analysis, as well as several of the EST clones,have three additional nucleotides (AAG) not found in Bbk, betweennucleotides 157 and 158 of Bbk. These nucleotides may be introduced asthe result of alternate splice site usage. The addition of these threenucleotides in the putative coding region maintains the predictedreading frame and would serve to introduce an arginine residue atposition 36. An analysis of protein databases using the putative Bbk ORFidentified sequences of only limited homology. Thus, novel proteins thatinteract with the apoptosis-related protein, Bak, can be identifiedthrough the use of the two hybrid system.

3. Expression of Bbk mRNA in fetal and adult tissues.

Northern blot analysis was performed to determine the expression patternand size of the Bbk messenger RNA. Northern blots of fetal and adulttissue probed with Bbk (FIG. 3) show that Bbk is a single mRNA speciesof approximately 0.8-1.2 kb. This message size is consistent with thenotion that a full length clone has been isolated. The northern blotanalysis also shows that Bbk is expressed in many diverse tissues with adistribution roughly similar to that of Bak (Kiefer, M. C., et al.,Nature 374:736 (1995)) supporting the belief that Bbk and Bak may becomplexed together in the cell.

4. Expression of Bbk in vitro and in transfected cells.

The deduced amino acid sequence of Bbk predicts a protein of MW 26.7 kD.The Bbk clone was subcloned from the yeast two-hybrid vector into pcDNA3(Clontech) for in vitro expression using the T7 promoter and formammalian expression using the human cytomegalovirus immediate earlypromoter. In order to detect the protein, the Bbk sequence was fused atthe amino terminus to a 14 amino acid segment derived from the influenzahemaglutinin antigen (HA). This short peptide provides a wellcharacterized epitope that permits immunological detection of the"tagged" protein by the monoclonal antibody 12CA5 (Boehringer Mannheim).In vitro transcription/translation of the Bbk clone in rabbitreticulocyte lysates produces a protein with electrophoretic mobility onSDS polyacrylamide gels corresponding to a molecular weight of about 37kD (FIG. 4A), in approximate agreement with the size predicted fromconceptual translation of the cDNA sequence plus the HA tag andnonspecific amino acids introduced by cloning. The pcDNA3 vectorexpressing HA-tagged Bbk was transfected into COS7 cells. Cell lysateswere prepared 48 hours after transfection, and analyzed by Western blotwith the anti-HA monoclonal antibody. The HA tagged Bbk protein, of theappropriate size, was detected in the COS7 cell extracts (FIG. 4B).These results demonstrate that the protein encoded by the isolated BbkcDNA can be expressed both in vitro and in vivo.

5. Expression of Bbk accelerates cell death in Rat1, HeLa, and BT549cells.

The HA-tagged Bbk clone expressed from the pcDNA3 vector wascotransfected with a plasmid expressing β-galactosidase into normal ratfibroblasts (Rat1 cells) and into human tumor lines HeLa and BT549, todetermine the effect of Bbk expression on cellular viability. Such acell death assay has been previously described (Miura, M., et al., Cell75:653 (1993); Boyd, J. M., et al., Oncogene 11:1921 (1995); Chittenden,T., et al., EMBO J. 14(22):5589 (1995); co-pending U.S. application Ser.No. 08/440,391, filed 12 May, 1995; and consists of co-transfection of agene of interest into cells with a β-galactosidase gene as a marker fortransfectants. The effect of transfected gene products upon cellularviability is measured by scoring blue cells (β-galactosidase positive)relative to vector controls cells 24 hours post-translection. Inert oranti-apoptotic gene products are manifested as flat (live) blue cells innumbers similar to those of the vector controls, while detrimental geneproducts can be seen as an overall reduction in blue cell numbers or anincrease in the frequency of round (dead) blue cells. The results inFIG. 5A clearly show a decrease in the number of blue cells when Rat1cells are transfected with a plasmid expressing Bbk. Thus, it appearsthat, like its binding partner Bak, Bbk can induce cell death. Both Bakand Bbk can induce apoptosis when expressed individually in Rat1 cells.While not intending to be bound by a particular theory, this suggestseither that the Bak/Bbk interaction is not necessary for the inductionof apoptosis or that there are rat homologs of Bak and Bbk that canfunctionally interact with the human proteins. Alternatively, theinteraction between these two proteins may serve a regulatory functionto modulate their apoptotic potential. The data in FIG. 5A demonstratethat the co-expression of Bak and Bbk does not block the induction ofapoptosis, suggesting that their ability to bind each other does notinhibit their ability to promote apoptosis. However, because Bak and Bbkeach is a potent promoter of cell death individually, it was notpossible in these assays to determine if their co-expression results ina cooperative induction of apoptosis.

The apoptotic function of Bbk can be reversed by the coexpression of theknown survival proteins, Bcl-2, Bcl-x_(L), and Epstein-Barr virus BHRF1(FIG. 5B). The extent of the cell death promoted by the apoptosisrelated proteins, Bak and Bik, is similarly reduced in the presence ofthe survival proteins (not shown). Increasing the ratio of theapoptosis-promoting protein, Bik, relative to these survival proteinscan restore Bik cytotoxicity. This observation suggests that in theappropriate setting apoptosis promoting proteins can actually repressthe action of survival proteins. The cell death promoting proteins maytherefore be used to induce apoptosis in cells where survival isdependent upon the action of one of the Bcl-2 related cell survivalproteins.

The ability of Bbk to induce apoptosis is also evident in the tumor celllines, HeLa and BT549 (FIG. 6A, B). The extent of apoptosis induced byBbk is comparable to that of Bak in the BT549 cells while in HeLa cellsBbk is somewhat less effective that Bak in the induction ot cell death.While Bbk can induce cell death, human tumor cells appear to displayvarying degrees of sensitivity to the apoptotic function of Bbk. Cellsthat are less sensitive to Bbk-induced apoptosis may, however, becomemore sensitive to conventional anti-cancer therapies subsequent totreatment with Bbk. Thus, a novel protein that binds Bak and inducesapoptosis in a variety of cell types has now been identified.

6. Bbk interacts with Bak, Bax, Bcl-x_(L) in vitro).

The reversal of Bbk induced apoptosis by cell survival members of theBcl-2 family may suggest that these proteins can interact with Bbk. Itwas desired to determine if Bbk interacts solely with Bak or it it couldalso bind to other proteins known to be involved in apoptosis. AGlutathione S-transferase (GST)-Bbk fusion protein was expressed in E.coli and purified over glutathione-agarose. HA-tagged Bak, Bax, and Bikas well as Bcl-x_(L) tagged with the FLAG epitope (Kodak) wereradioactively labeled by in vitro translation and subsequently incubatedwith GST-Bbk or GST alone. Complexes were isolated onglutathione-agarose and analyzed by SDS-PAGE. FIG. 7 clearly shows Bbkinteraction with Bak, BCl-X_(L), and Bax but not with Bik. Theseinteractons are specific, as the GST alone fails to complex with any ofthe in vitro translated material. Thus it appears that Bbk can interactwith several members of the Bcl-2 family. Bbk does not appear tointeract exclusively with the cell death inducing members of the Bcl-2family, as demonstrated by it's interaction with Bcl-X_(L), despite thefact that Bbk was isolated by virtue of its interaction with the celldeath promoter, Bak. It is interesting to note that Bbk cannot, however,interact with Bik, a novel death inducing protein that shares the BH3domain with Bcl-2 family members (Boyd, J. M., et al., Oncogene 11:1921(1995)).

7. Bbk shares sequence homology with the Bcl-2 protein family.

The observations that Bbk interacts with several members of the Bcl-2family and that it shares apoptosis related function suggested that Bbkmight additionally share sequence homology. The database searchesperformed above did not reveal any sequence homology to Bcl-2 familymembers, however, a careful visual inspection identified a motif that ishighly homologous to the BH2 domain of Bcl-2 and related family members(FIG. 8A). Alignment of the Bbk ORF with Bcl-2 family members using, theBH2 domain as an anchor did not reveal any homology to the BH1 domain ofBcl-2 but does show some homology (FIG. 8B) to the newly defined BH3domain (co-pending U.S. application Ser. No. 08/440,391, filed 12 May,1995, U.S. Pat. No. 5,656,725 (BH3 is referred to therein as the GDDomain)). Thus, Bbk appears to be a novel death-promoting member of theBcl-2 protein family.

8. Bbk induced apoptosis is mediated by its BH3-like domain

It has been previously shown that the BH3 domain of Bak is bothnecessary and sufficient for its induction of cell death (co-pendingU.S. application Ser. No. 08/440,391, filed 12 May, 1995, U.S. Pat. No.5,656,725 (BH3 is referred to therein as the GD Domain)). Because Bbkshares a weak homology to the Bak BH3 domain, it was desired todetermine whether this region provides a similar function for Bbk. Totest this theory ai series of deletions that encroach upon the putativeBH3 domain were made and tested in the Rat-1 cell death assay. Theresults in FIG. 9A show that two of these mutants, Δ1-105 (a deletion ofthe N-terminus to amino acid residue 106) and Δ142-249 (a deletion ofthe C-terminus beginning at amino acid residue 142), still retain theability to induce apoptosis. These results suggest that a large portionof the Bbk molecule (including the BH2 domain) is dispensible for thecytotoxic activity of Bbk. Further, these data define the region betweenamino acids 106 to 141 as necessary for Bbk to induce cell death.Interestingly, this region coincides with the putative Bbk BH3 domain(residues 125-137). To definitively prove that this region was requiredfor Bbk cytotoxicity, four alanine scanning mutants (FIG. 9B) thatmutate several conserved residues were made and tested for their abilityto induce apoptosis in Ratl cells . The results shown in FIG. 9Cdemonstrate that PM-LVLEE, which contains five alamine substitutions inthe BH3 element, has completely lost its apoptosis inductioncapabilities. To prove that the abolition of cytotoxicity in PM-LVLEE isnot due to a failure to be synthesized, its expression was verified inCOS7 cells where it was produced at levels comparable to those of wildtype Bbk. The data therefore support the conclusion that the BH3 regionof Bbk is absolutely necessary for its apoptotic function. The remainingmutants, PM-V, PM-L and PM-EE, are sin-le and double alanine mutationsthat were constructed to more precisely define the BH3 residues requiredfor Bbk-induced apoptosis. The data in FIG. 9C demonstrate that each ofthese substitutions reduces, but does not abolish, the induction of celldeath, indicating that all of the mutated residues are required in partfor Bbk cytotoxicity. Again, each of these mutants was expressed in COS7cells at levels comparable to those of wild type Bbk.

To determine if the Bbk BH3 domain was also sufficient to induceapoptosis, a plasmid expressing only amino acids 117-166 of Bbk (whichincludes the Bbk BH3 domain between residues 125-137) was transfectedinto Rat1 cells. The results in FIG. 10 show that expression of this 50amino acid peptide encompassing the Bbk BH3 domain is sufficient toinduce apoptosis. Thus, it appears that the BH3 domain of Bbk isanalogous in function to the Bak BH3 domain, in that it is bothnecessary and sufficient for the induction of apoptosis.

9. Apoptosis induction by Bbk correlates with its ability to bind Bak.

Co-pending U.S. application Ser. No. 08/440.391, filed 12 May, 1995,U.S. Pat. No. 5,656,725 demonstrated that the Bak BH3 domain isresponsible for its cytotoxic activity as well as its ability to bindthe Bcl-x_(L), an anti-apoptotic member of the Bcl-2 fanily. Since theBbk BH3 domain appears to share the ability to induce apoptosis functionin a manner similar to that of the Bak BH3 domain, it was of interest todetermine whether the Bbk BH3 domain mediates the Bbk/Bak interaction.To test this possibility, the alanine mutations used above to define theapoptosis inducing domain of Bbk (PM-LVLEE, PM-V, PM-L, PM-EE) werefused to the Gal4 activation domain for use in the yeast two hybridsystem with Bak as bait. In this assay, the expression levels ofβ-galactosidase are a measure of the affinity and/or stability ofinteraction between the two proteins, with high levels indicating astrong interaction and low levels indicating a weak interaction. FIG. 11shows that PM-LVLEE, the Bbk mutant that has lost the ability to induceapoptosis, has also lost the ability to bind to Bak, as demonstrated bylow levels of β-galactosidase (comparable to the negative control). Themutants that have retained intermediate levels of apoptotic potential(PM-V, PM-L, PM-EE) retain intermediate levels of interaction with Bak.This direct correlation between Bbk cytotoxicity and Bbk binding to Bakfurther supports the hypothesis that the Bbk/Bak interaction is requiredfor the induction of apoptosis. Alternatively, there remains thepossibility that there are additional binding partners for Bbk that alsointeract with its BH3 domain to effect cell death.

All publications mentioned in this specification are herein incorporatedby reference, to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.It will be understood that the invention is capable of furthermodifications and this application is intended to cover any variations,uses, or adoptions of the invention including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains, and is intended to be limited onlyby the appended claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 29                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino acid                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       LeuArgArgLeuValAlaLeuLeuGluGluGluAlaGlu                                       510                                                                           (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino acid                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       LeuArgArgLeuAlaAlaLeuLeuGluGluGluAlaGlu                                       510                                                                           (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       LeuArgArgLeuValAlaLeuAlaGluGluGluAlaGlu                                       510                                                                           (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       LeuArgArgLeuValAlaLeuLeuGluAlaAlaAlaGlu                                       510                                                                           (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       LeuArgArgLeuValAlaLeuLeuGluGluGluAlaGlu                                       510                                                                           (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       LeuArgArgLeuAlaAlaLeuLeuGluGluGluAlaGlu                                       510                                                                           (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       LeuArgArgLeuValAlaLeuAlaGluGluGluAlaGlu                                       510                                                                           (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       LeuArgArgLeuValAlaLeuLeuGluAlaAlaAlaGlu                                       510                                                                           (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 958 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       CGCAAGTTGAGTGGAGGAGGCGGCGGTGGGGCCCCGGACCAGGTGCCTCCATGGCAGGCT60                CTGAAGAGCTGGGGCTCCGGGAAGACACGCTGAGGGTCCTAGCTGCCTTCCTTAGGCGTG120               GTGAGGCTGCCGGGTCTCCTGTTCCAACTCCACCTAGCCCTGCCCAAGAAGAGCCAACAG180               ACTTCCTGAGCCGCCTTCGAAGATGTCTTCCCTGCTCCCTGGGGCGAGGAGCAGCCCCCT240               CTGAGTCCCCTCGGCCTTGCTCTCTGCCCATCCGCCCCTGCTATGGTTTAGAGCCTGGCC300               CAGCTACTCCAGACTTCTATGCTTTGGTGGCCCAGCGGCTGGAACAGCTGGTCCAAGAGC360               AGCTGAAATCTCCGCCCAGCCCAGAATTACAGGGTCCCCCATCGACAGAGAAGGAAGCCA420               TACTGCGGAGGCTGGTGGCCCTGCTGGAGGAGGAGGCAGAAGTCATTAACCAGAAGCTGG480               CCTCGGACCCCGCCCTGCGCAGCAAGCTGGTCCGCCTGTCCTCCGACTCTTTCGCCCGCC540               TGGTGGAGCTGTTCTGTAGCCGGGATGACAGCTCTCGCCCAAGCCGAGCATGCCCCGGGC600               CCCCGCCTCCTTCCCCGGAGCCCCTGGCCCGCCTGGCCCTAGCCATGGAGCTGAGCCGGC660               GCGTGGCCGGGCTGGGGGGCACCCTGGCCGGACTCAGCGTGGAGCACGTGCACAGCTTCA720               CGCCCTGGATCCAGGCCCACGGGGGCTGGGAGGGCATCCTGGCTGTTTCACCCGTGGACT780               TGAACTTGCCATTGGACTGAGCTCTTTCTCAGAAGCTGCTACAAGATGACACCTCATGTC840               CCTGCCCTCTTCGTGTGCTTTTCCAAGTCTTCCTATTCCACTCAGGGCTGTGGGGTGGTG900               GTTGCCCTACCTGTTTTTGCCAAAAATAAATTGTTTAAAACTTTTCTTATTAAAAACG958                 (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 958 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      GCGTTCAACTCACCTCCTCCGCCGCCACCCCGGGGCCTGGTCCACGGAGGTACCGTCCGA60                GACTTCTCGACCCCGAGGCCCTTCTGTGCGACTCCCAGGATCGACGGAAGGAATCCGCAC120               CACTCCGACGGCCCAGAGGACAAGGTTGAGGTGGATCGGGACGGGTTCTTCTCGGTTGTC180               TGAAGGACTCGGCGGAAGCTTCTACAGAAGGGACGAGGGACCCCGCTCCTCGTCGGGGGA240               GACTCAGGGGAGCCGGAACGAGAGACGGGTAGGCGGGGACGATACCAAATCTCGGACCGG300               GTCGATGAGGTCTGAAGATACGAAACCACCGGGTCGCCGACCTTGTCGACCAGGTTCTCG360               TCGACTTTAGAGGCGGGTCGGGTCTTAATGTCCCAGGGGGTAGCTGTCTCTTCCTTCGGT420               ATGACGCCTCCGACCACCGGGACGACCTCCTCCTCCGTCTTCAGTAATTGGTCTTCGACC480               GGAGCCTGGGGCGGGACGCGTCGTTCGACCAGGCGGACAGGAGGCTGAGAAAGCGGGCGG540               ACCACCTCGACAAGACATCGGCCCTACTGTCGAGAGCGGGTTCGGCTCGTACGGGGCCCG600               GGGGCGGAGGAAGGGGCCTCGGGGACCGGGCGGACCGGGATCGGTACCTCGACTCGGCCG660               CGCACCGGCCCGACCCCCCGTGGGACCGGCCTGAGTCGCACCTCGTGCACGTGTCGAAGT720               GCGGGACCTAGGTCCGGGTGCCCCCGACCCTCCCGTAGGACCGACAAAGTGGGCACCTGA780               ACTTGAACGGTAACCTGACTCGAGAAAGAGTCTTCGACGATGTTCTACTGTGGAGTACAG840               GGACGGGAGAAGCACACGAAAAGGTTCAGAAGGATAAGGTGAGTCCCGACACCCCACCAC900               CAACGGGATGGACAAAAACGGTTTTTATTTAACAAATTTTGAAAAGAATAATTTTTGC958                 (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 249 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      MetAlaGlySerGluGluLeuGlyLeuArgGluAspThrLeuArg                                 51015                                                                         ValLeuAlaAlaPheLeuArgArgGlyGluAlaAlaGlySerPro                                 202530                                                                        ValProThrProProSerProAlaGlnGluGluProThrAspPhe                                 354045                                                                        LeuSerArgLeuArgArgCysLeuProCysSerLeuGlyArgGly                                 505560                                                                        AlaAlaProSerGluSerProArgProCysSerLeuProIleArg                                 657075                                                                        ProCysTyrGlyLeuGluProGlyProAlaThrProAspPheTyr                                 808590                                                                        AlaLeuValAlaGlnArgLeuGluGlnLeuValGlnGluGlnLeu                                 95100105                                                                      LysSerProProSerProGluLeuGlnGlyProProSerThrGlu                                 110115120                                                                     LysGluAlaIleLeuArgArgLeuValAlaLeuLeuGluGluGlu                                 125130135                                                                     AlaGluValIleAsnGlnLysLeuAlaSerAspProAlaLeuArg                                 140145150                                                                     SerLysLeuValArgLeuSerSerAspSerPheAlaArgLeuVal                                 155160165                                                                     GluLeuPheCysSerArgAspAspSerSerArgProSerArgAla                                 170175180                                                                     CysProGlyProProProProSerProGluProLeuAlaArgLeu                                 185190195                                                                     AlaLeuAlaMetGluLeuSerArgArgValAlaGlyLeuGlyGly                                 200205210                                                                     ThrLeuAlaGlyLeuSerValGluHisValHisSerPheThrPro                                 215220225                                                                     TrpIleGlnAlaHisGlyGlyTrpGluGlyIleLeuAlaValSer                                 230235240                                                                     ProValAspLeuAsnLeuProLeuAsp                                                   245                                                                           (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      TrpIleGlnAlaHisGlyGlyTrpGluGlyIleLeuAlaVal                                    510                                                                           (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      TrpIleAlaGlnArgGlyGlyTrpValAlaAlaLeuAsnLeu                                    510                                                                           (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      TrpIleGlnAspGlnGlyGlyTrpAspGlyLeuLeuSerTyr                                    510                                                                           (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      TrpArgSerProAsnProGlySerTrpValSerCysGluGlnVal                                 51015                                                                         (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      TrpIleGlnAspAsnGlyGlyTrpAspAlaPheValGluLeu                                    510                                                                           (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      TrpIleGlnGluAsnGlyGlyTrpAspThrPheValGluLeu                                    510                                                                           (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      LeuArgArgLeuValAlaLeuLeuGluGluGluAlaGlu                                       510                                                                           (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      ValGlyArgGlnLeuAlaIleIleGlyAspAspIleAsn                                       510                                                                           (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      LeuSerGluCysLeuLysArgIleGlyAspGluLeuAsp                                       510                                                                           (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      LeuAlaLeuArgLeuAlaCysIleGlyAspGluMetAsp                                       510                                                                           (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      ValHisLeuAlaLeuArgGlnAlaGlyAspAspPheSer                                       510                                                                           (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      ValLysGlnAlaLeuArgGluAlaGlyAspGluPheGlu                                       510                                                                           (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      LeuArgArgLeuValAlaLeuLeuGluGluGluAlaGlu                                       510                                                                           (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      AlaArgArgLeuAlaAlaLeuAlaGluAlaAlaAlaGlu                                       510                                                                           (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      LeuArgArgLeuAlaAlaLeuLeuGluGluGluAlaGlu                                       510                                                                           (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      LeuArgArgLeuValAlaLeuAlaGluGluGluAlaGlu                                       510                                                                           (2) INFORMATION FOR SEQ ID NO:28:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                      LeuArgArgLeuValAlaLeuLeuGluAlaAlaAlaGlu                                       510                                                                           (2) INFORMATION FOR SEQ ID NO:29:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                      MetGlyTyrProTyrAspValProAspTyrAlaSerLeuSer                                    510                                                                           __________________________________________________________________________

What is claimed is:
 1. An isolated Bbk protein comprising the amino acidsequence set forth as SEQ ID NO:11.
 2. A composition comprising the Bbkprotein of claim 1 and a carrier.
 3. A method for inducing apoptosis ina nonapoptotic cell in vitro comprising expressing in said cell anamount of the Bbk protein of claim 1 effective to induce apoptosis insaid cell.
 4. An isolated peptide comprising the Bbk RH3 domain, whereinsaid domain comprises an amino acid sequence selected from the groupconsisting of the sequences set forth as SEQ ID NOS:5-8.
 5. The peptideof claim 4 wherein said peptide consists of an amino acid sequenceselected from the group consisting of:LRRLVALLEEEAE (SEQ ID NO:5),LRRLAALLEEEAE (SEQ ID NO:6), LRRLVALAEEEAE (SEQ ID NO:7), andLRRLVALLEAAAE (SEQ ID NO:8).